Her2-targeting molecules comprising de-immunized, shiga toxin a subunit scaffolds

ABSTRACT

Provided herein are HER2-targeting molecules comprising Shiga toxin A Subunit derived polypeptides having 1) de-immunization and 2) reduced, protease-cleavage sensitivity while retaining Shiga toxin function(s), such as, e.g., potent cytotoxicity via ribosome inhibition. Certain HER2-targeting molecules of the present invention exhibit reduced immunogenic potential in mammals and are well-tolerated by mammals while retaining aforementioned features. The HER2-targeting molecules of the present invention have uses for selectively killing specific cells (e.g., HER positive tumor cells); for selectively delivering cargos to specific cells (e.g., HER positive tumor cells), and as therapeutic and/or diagnostic molecules for treating and diagnosing a variety of conditions, including cancers and tumors involving the expression or over-expression of cell-surface HER2.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0.1] This application is a Continuation of U.S. Application No.17/543,207, filed Dec. 6, 2021, which is a Continuation of U.S.Application No. 17/072,562, filed Oct. 16, 2020, (issued as U.S. Pat.No. 11,225,509), which is a Continuation of International ApplicationNo. PCT/US2019/027627, filed Apr. 16, 2019, which claims benefit under35 U.S.C. §119(e) to U.S. Provisional Pat. Application No. 62/659,116,filed Apr. 17, 2018, the contents of each of which are incorporatedherein by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(MTEM_022_03US_SeqList_ST26.xml; Size: 277,044 bytes; and Date ofCreation: Feb. 14, 2023) are herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to HER2-targeting molecules comprisingShiga toxin effector polypeptides, derived from the A Subunits ofnaturally occurring Shiga toxins, that comprise a combination ofmutations providing (1) de-immunization, (2) a reduction in proteasesensitivity, and/or (3) an embedded, T-cell epitope(s); wherein theShiga toxin effector polypeptides retain one or more Shiga toxinfunctions, such as, e.g., potent cytotoxicity. The HER2-targetingmolecules of the present invention are useful for administration tomulticellular organisms, such as, e.g., when it is desirable to (1)eliminate or reduce non-specific toxicities and/or (2) eliminate orreduce certain immune responses. The HER2-targeting molecules of thepresent invention are useful (1) for selectively killing specificHER2-positive cell type(s) amongst other cells and (2) as therapeuticmolecules for treating a variety of diseases, disorders, and conditionsinvolving HER2-expressing cells, including cancers and tumors.

BACKGROUND

HER2 is a particularly attractive molecular target for therapeuticsbecause of its overexpression on the surfaces of tumor and/or cancercells, its correlation with poor prognoses, and its functional roles intumorigenesis and cancer development, such as invasiveness andmetastasis, and anti-neoplastic drug resistance (Nielsen D et al.,Breast 22: 1-12 (2013); Ocaña A, Pandiella A, Curr Pharm Des 19: 808-17(2013)).

HER2 (human epidermal growth factor receptor 2) is a type Itransmembrane tyrosine kinase receptor of the ErbB family (Yamamoto T etal., Nature 319: 230-4 (1986); Slamon D et al., Science 235: 177-82(1987)). Members of the ErbB family are integral glycoproteins whichregulate cell growth, differentiation, and survival by binding to growthfactor ligands as dimers (Chantry A, J Biol Chem 270: 3068-73 (1995)).

HER2 is prominently associated with the pathogenesis, progression, andprognosis of certain breast cancers, among other cancers (Citri A,Yarden Y, Nat Rev Mol Cell Biol 7: 505-16 (2006)). The proto-oncogeneHER2, which encodes HER2, was found to be amplified and overexpressed inbreast cancer cells (King et al., Science 229: 974-6 (1985); Slamon etal. Science 235: 177-82 (1987)). Amplification and/or over-expression ofHER2 occurs in approximately 15-30% of breast cancers, and the presenceof HER2 in breast cancer is strongly associated with aggressivemalignancy, increased disease recurrence, and poor prognosis (Slamon Det al., Science 244: 707-12 (1989)); Bernstein H, N Engl JMed 353:1652-4 (2005); Pritchard I et al., N EnglJMed 354: 2103-11 (2006); TanM, Yu D, Adv Exp Med Biol 608: 119-29 (2007); Mitri Z et al., ChemotherRes Pract 2012: 743193 (2012)).

HER2 is overexpressed in many other diverse cancers and may functionallycontribute to tumorigenesis generally. HER2 overexpression has beenobserved in breast, colorectal, endometrial, esophageal, gastric, headand neck, lung, ovarian, prostate, pancreatic, and testicular germ celltumor cells (Kern J et al., Cancer Res 50: 5184-7 (1990); Natali P etal., Int J Cancer 45: 457-61 (1990); Jaehne J et al., J Cancer Res ClinOncol 118: 474-9 (1992); Signoretti S et al., J Natl Cancer Inst 92:1918-25 (2000); Di Lorenzo G et al., Clin Cancer Res 8: 3438-44 (2002);Owens M et al., Clin Breast Cancer 5: 63-9 (2004); Roskoski R, BiochemBiophys Res Commun 319: 1-11 (2004); Cohen G et al., Cancer Res 66:5656-64 (2006); Santin A et al., Int J Gynaecol Obstet 102: 128-31(2008); Vermeij J et al., BMC Cancer 8: 3 (2008); Chen P et al., J ClinPathol 66: 113-9 (2013); Chou et al., Genome Med 5: 78 (2013); Cros J etal., Ann Oncol 24: 2624-9 (2013); König A et al., Anticancer Res 33:4975-82 (2013); Sugishita Y et al., Int J Oncol 42: 1589-96 (2013)). Inaddition, overexpression ofHER2 in a tumor cell can confer drugresistance to anti-neoplastic agents (Koutras A et al., Crit Rev OncolHematol 74: 73-8 (2010)).

There is an urgent need for new therapeutics to supplement present daytherapies for HER2-bearing neoplasms. Thus, it would be desirable tohave cytotoxic cell-targeting molecules which target HER2 for use astherapeutic molecules to treat a variety of diseases, such as, e.g.,cancers and tumors, that can be treated by selective killing of, orselective delivery of a beneficial agent into, a HER2 positive cell. Inparticular, it would be desirable to have HER2-binding, cytotoxic,cell-targeting molecules exhibiting low antigenicity and/orimmunogenicity, low off-target toxicity, and potent cytotoxicity.Furthermore, it would be desirable to have HER2-targeting therapeuticand/or diagnostic molecules exhibiting low antigenicity and/orimmunogenicity, low off-target toxicity, high stability, and/or theability to deliver peptide-epitope cargos for presentation by the MHCclass I system of a target cell. For example, it would be desirable tohave cytotoxic HER2-targeting molecules comprising Shiga toxin A Subunitderived components which maintain potent cytotoxicity to target cellswhile 1) reducing the potential for unwanted antigenicities and/orimmunogenicities, 2) reducing the potential for non-specific toxicities,3) allowing for drug tolerability over a wide range of dosages, 4)allowing for drug tolerance after repeated administration, and 5)retaining effectiveness in the presence of one or more additionalHER2-targeted therapies.

SUMMARY OF THE INVENTION

The Shiga toxin A Subunit derived components of the HER2-targetingmolecules of the present invention each comprise a combination offeatures (e.g., de-immunized sub-region(s) and a protease-cleavageresistant sub-region). Certain combination Shiga toxin effectorpolypeptides of the present invention are more useful because theyprovide several Shiga toxin effector functions in a single polypeptide,such as, e.g., promoting cellular internalization, directingsub-cellular routing to the cytosol, ribosome inactivation, and/ordelivering cargos to subcellular compartments. Certain HER2-targetingmolecules of the present invention are more useful because they providea combination of several properties in a single molecule, such as, e.g.,efficient cellular internalization, potent cell-targeted cytotoxicity,selective cytotoxicity, de-immunization, low non-specific toxicity athigh dosages, high stability, CD8+ T-cell hyper-immunization, and/or 5)retention of effectiveness in the presence of one or more additionalHER2-targeted therapies. Different embodiments of the HER2-targetingmolecules of the present invention are described below with reference tosets of embodiments numbered #1-3.

Embodiment Set #1 - HER2-Targeting Molecule Comprising a De-ImmunizedShiga Toxin Effector Polypeptide Comprising an Embedded or Inserted,Heterologous, T-Cell Epitope and a Non-Overlapping De-ImmunizedSub-Region

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule (HER2/neu/ErbB2) and (ii) a de-immunized, Shiga toxinA Subunit effector polypeptide. For example, certain embodiments of Set#1 is the cell-targeting molecule comprising (i) a binding regioncapable of specifically binding an extracellular target biomolecule and(ii) a de-immunized, Shiga toxin effector polypeptide comprising a Shigatoxin A1 fragment region and a carboxy-terminus, wherein the Shiga toxinA subunit effector polypeptide comprises: (a) at least one inserted orembedded, heterologous epitope; and (b) at least one disrupted,endogenous, B-cell and/or CD4+ T-cell epitope region which does notoverlap with the embedded or inserted, heterologous, T-cell epitope. Forcertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. The Shiga toxin effectorpolypeptide of Embodiment Set #1 may be truncated at itscarboxy-terminus, relative to a wild-type Shiga toxin A Subunit,resulting in the elimination of one or more endogenous, B-cell and/orCD4+ T-cell epitope regions. The Shiga toxin effector polypeptide ofEmbodiment Set # 1 may comprise a disrupted furin-cleavage motif at thecarboxy-terminus of the A1 fragment region. In certain embodiments, thefurin-cleavage motif is disrupted by a carboxy-terminal truncation ofthe Shiga toxin effector polypeptide as compared to the carboxy-terminusof a wild-type Shiga toxin A Subunit. For example, the present inventionprovides a Shiga toxin effector polypeptide comprising a Shiga toxin A1fragment region and a carboxy-terminus, wherein the Shiga toxin Asubunit effector polypeptide comprises: (a) an embedded or inserted,heterologous, epitope; (b) a disruption of at least one, endogenous,B-cell and/or CD4+ T-cell epitope region; and (c) a disruptedfurin-cleavage motif at the carboxy-terminus of the Shiga toxin A1fragment region; wherein the Shiga toxin A subunit effector polypeptideis capable of exhibiting a Shiga toxin effector function. In a furtherexample, the present invention provides a Shiga toxin A subunit effectorpolypeptide comprising a Shiga toxin A1 fragment region and acarboxy-terminus, wherein the Shiga toxin A subunit effector polypeptidecomprises (a) an embedded or inserted, heterologous, CD8+ T-cell epitopewhich disrupts an endogenous, B-cell and/or CD4+ T-cell epitope region;(b) a disruption of at least four, endogenous, B-cell and/or CD4+ T-cellepitope regions which do not overlap with the embedded or inserted,heterologous, CD8+ T-cell epitope; and (c) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment region; andwherein the Shiga toxin A subunit effector polypeptide is truncated atits carboxy-terminus, relative to a wild-type Shiga toxin A subunit,resulting in the elimination of one or more endogenous, B-cell and/orCD4+ T-cell epitope regions; wherein the Shiga toxin A subunit effectorpolypeptide is capable of exhibiting a Shiga toxin effector function.Accordingly, the present invention provides a HER2-targeting moleculethat comprises: (i) an immunoglobulin binding region capable ofspecifically binding an extracellular part of HER2/neu/ErbB2, andcomprising one or more of: an antibody variable fragment, asingle-domain antibody fragment, a single-chain variable fragment, a Fdfragment, an antigen-binding fragment, an autonomous VH domain, a V_(H)Hfragment derived from a camelid antibody, a heavy-chain antibody domainderived from a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor; and ii) a Shiga toxin A subuniteffector polypeptide comprising a Shiga toxin A1 fragment region and acarboxy-terminus, wherein the Shiga toxin A subunit effector polypeptidecomprises: (a) an embedded or inserted, heterologous, CD8+ T-cellepitope which disrupts an endogenous, B-cell and/or CD4+ T-cell epitoperegion; (b) a disruption of at least four, endogenous, B-cell and/orCD4+ T-cell epitope regions which do not overlap with the embedded orinserted, heterologous, CD8+ T-cell epitope; and (c) a disruptedfurin-cleavage motif at the carboxy-terminus of the Shiga toxin A1fragment region; and wherein the Shiga toxin A subunit effectorpolypeptide is truncated at its carboxy-terminus, relative to awild-type Shiga toxin A subunit, resulting in the elimination of one ormore endogenous, B-cell and/or CD4+ T-cell epitope regions; wherein theShiga toxin A subunit effector polypeptide is capable of exhibiting aShiga toxin effector function. For certain further embodiments, thecell-targeting molecule is capable when introduced to cells ofexhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a second cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide components comprise a wild-type Shiga toxin furin-cleavagesite at the carboxy terminus of its A1 fragment region.

For certain embodiments of Embodiment Set # 1, the cell-targetingmolecule exhibits reduced relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., awild-type Shiga toxin A Subunit or a third cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment.

In certain embodiments of Embodiment Set # 1, the binding region andShiga toxin effector polypeptide are linked together, either directly orindirectly.

In certain embodiments of Embodiment Set #1, the binding regioncomprises a polypeptide comprising an immunoglobulin orimmunoglobulin-type binding region. In certain further embodiments, thebinding region comprising a polypeptide selected from the groupconsisting of: an autonomous V_(H) domain, single-domain antibodyfragment (sdAb), nanobody®, heavy chain-antibody domain derived from acamelid antibody (V_(H)H or V_(H) domain fragment), heavy-chain antibodydomain derived from a cartilaginous fish antibody (V_(H)H or V_(H)domain fragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality. In certainembodiments, the binding region comprises a polypeptide selected fromthe group consisting of: an autonomous V_(H) domain, single-domainantibody fragment (sdAb), nanobody®, heavy chain-antibody domain derivedfrom a camelid antibody (V_(H)H or V_(H) domain fragment), heavy-chainantibody domain derived from a cartilaginous fish antibody (V_(H)H orV_(H) domain fragment), immunoglobulin new antigen receptor (IgNAR),V_(NAR) fragment, single-chain variable fragment (scFv), antibodyvariable fragment (Fv), Fd fragment, and antigen-binding fragment (Fab).In certain embodiments, the cell-targeting molecule of the presentinvention comprises an immunoglobulin binding region capable ofspecifically binding an extracellular part of HER2/neu/ErbB2, andcomprising one or more of: an antibody variable fragment, asingle-domain antibody fragment, a single-chain variable fragment, a Fdfragment, an antigen-binding fragment, an autonomous VH domain, a V_(H)Hfragment derived from a camelid antibody, a heavy-chain antibody domainderived from a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor. In certain embodiments, the bindingregion comprises, consists essentially of, or consists of a single-chainvariable fragment (scFv). In certain embodiments, the binding regioncomprises a single-chain variable fragment (scFv). In certainembodiments, the binding region comprises, consists essentially of, orconsists of a V_(H)H fragment derived from a camelid antibody.

In certain embodiments of Embodiment Set # 1, the binding region and theShiga toxin effector polypeptide are fused, either directly orindirectly, forming a continuous polypeptide such that the bindingregion is associated, either directly or indirectly, with thecarboxy-terminus of the Shiga toxin effector polypeptide.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Set # 1, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is (i) capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule, such as, e.g., athird cell-targeting molecule consisting of the cell-targeting moleculeexcept for all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment, and/or (ii) capable ofexhibiting a significant level of intracellular routing activity to theendoplasmic reticulum and/or cytosol from an endosomal starting locationof a cell.

For certain embodiments of Embodiment Set # 1, whereby administration ofthe cell-targeting molecule of the present invention to a cellphysically coupled with the extracellular target biomolecule of thecell-targeting molecule’s binding region, the cell-targeting molecule iscapable of causing death of the cell. For certain further embodiments,administration of the cell-targeting molecule of the invention to twodifferent populations of cell types which differ with respect to thepresence or level of the extracellular target biomolecule, thecell-targeting molecule is capable of causing cell death to thecell-types physically coupled with an extracellular target biomoleculeof the cytotoxic cell-targeting molecule’s binding region at a CD₅₀ atleast three times or less than the CD₅₀ to cell types which are notphysically coupled with an extracellular target biomolecule of thecell-targeting molecule’s binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first populations of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule’s binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule’sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule’s binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Set # 1, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity. For certain furtherembodiments, the cell-targeting molecule exhibits reduced relativeantigenicity and/or relative immunogenicity as compared to a referencemolecule, such as, e.g., a wild-type Shiga toxin A Subunit or a thirdcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment.

In certain embodiments of Embodiment Set #1, the heterologous, T-cellepitope is a CD8+ T-cell epitope, such as, e.g., with regard to a humanimmune system. For certain further embodiments, the heterologous, T-cellepitope is capable of being presented by a MHC class I molecule of acell. In certain further embodiments, the cell-targeting molecule of thepresent invention is capable of one or more the following: entering acell, inhibiting a ribosome function, causing cytostasis, causing celldeath, and/or delivering the embedded or inserted, heterologous, T-cellepitope to a MHC class I molecule for presentation on a cellularsurface. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a thirdcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment.

For certain embodiments of Embodiment Set # 1, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a fourth cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage siteat the carboxy terminus of its A1 fragment region. In certain furtherembodiments, the Shiga toxin effector polypeptide is not cytotoxic andthe molecular moiety is cytotoxic.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculecomprises a molecular moiety associated with the carboxy-terminus of theShiga toxin effector polypeptide. In certain embodiments, the molecularmoiety comprises or consists of the binding region. In certainembodiments, the molecular moiety comprises at least one amino acid andthe Shiga toxin effector polypeptide is linked to at least one aminoacid residue of the molecular moiety. In certain further embodiments,the molecular moiety and the Shiga toxin effector polypeptide are fusedforming a continuous polypeptide.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculefurther comprises a cytotoxic molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. For certainembodiments, the cytotoxic molecular moiety is a cytotoxic agent, suchas, e.g., a small molecule chemotherapeutic agent, anti-neoplasticagent, cytotoxic antibiotic, alkylating agent, antimetabolite,topoisomerase inhibitor, and/or tubulin inhibitor known to the skilledworker and/or described herein. For certain further embodiments, thecytotoxic molecular moiety is cytotoxic at concentrations of less than10,000, 5,000, 1,000, 500, or 200 pM.

In certain embodiments of Embodiment Set # 1, the binding region islinked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is an immunoglobulin orimmunoglobulin-type binding region.

In certain embodiments of Embodiment Set #1, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned (1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ IDNO: 1), Shiga toxin (SEQ ID NO:2), or another Shiga toxin 1 variantsequence (e.g. SEQ ID NOs: 4-6); or (2) at 247-250 of the A Subunit ofShiga-like toxin 2 (SEQ ID NO:3) or a Shiga-like toxin 2 variantsequence (e.g. SEQ ID NOs: 7-18), or the equivalent amino acid sequenceposition in any Shiga toxin A Subunit. In certain further embodiments,the mutation is an amino acid residue substitution of an arginineresidue with a non-positively charged, amino acid residue.

In certain embodiments of Embodiment Set # 1, the Shiga toxin effectorpolypeptide comprises, consists essentially of, or consists of: (i)amino acids 75 to 251 of any one of SEQ ID NOs: 1-6 and 37; (ii) aminoacids 1 to 241 of any one of SEQ ID NOs: 1-18 and 75-89; (iii) aminoacids 1 to 251 of any one of SEQ ID NOs: 1-6, 37, and 75-89; or (iv)amino acids 1 to 261 of any one of SEQ ID NOs: 1-3; wherein the Shigatoxin effector polypeptide comprises at least one embedded or inserted,heterologous T-cell epitope and at least one (two, three, four or more)disrupted, endogenous, B-cell and/or CD4+ T-cell epitope region(s) whichdoes not overlap with the embedded or inserted, heterologous, T-cellepitope.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a third cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment.

In certain embodiments of Embodiment Set #1, the binding region maycomprise at least one heavy-chain variable domain polypeptide comprising(i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ IDNOs: 51, SEQ ID NO:52, and SEQ ID NO:53, respectively; and at least onelight-chain variable domain polypeptide comprising: (i) the LCDR1,LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:54, SEQ IDNO:55, and SEQ ID NO:56, respectively. For example, the binding regionmay comprises at least one heavy-chain variable domain polypeptidecomprising (i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown inSEQ ID NOs: 57, SEQ ID NO:58, and SEQ ID NO:59, respectively; and atleast one light-chain variable domain polypeptide comprising (i) theLCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:60, SEQID NO:61, and SEQ ID NO:62, respectively. For example, the bindingregion may comprises at least one heavy-chain variable domainpolypeptide comprising (i) the HCDR1, HCDR2, and HCDR3 amino acidsequences shown in SEQ ID NOs: 63, SEQ ID NO:64, and SEQ ID NO:65,respectively; and at least one light-chain variable domain polypeptidecomprising (i) the LCDR1, LCDR2, and LCDR3 amino acid sequences shown inSEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively. The bindingregion having these CDRs may be an immunoglobulin binding regioncomprising a single-chain variable fragment.

In certain embodiments of Embodiment Set #1, the binding region maycomprises the binding region comprises a polypeptide selected from thegroup consisting of: a) a heavy chain only variable (V_(H)H) domaincomprising (i) a HCDR1 comprising or consisting essentially of the aminoacid sequences as shown in SEQ ID NO:69 or SEQ ID NO:72; (ii) a HCDR2comprising or consisting essentially of the amino acid sequence as shownin SEQ ID NO:70 or SEQ ID NO:73; and (iii) a HCDR3 comprising orconsisting essentially of the amino acid sequence as shown in SEQ IDNO:71 or SEQ ID NO:74. The binding region having these CDRs may be animmunoglobulin binding region comprising a heavy chain only variable(V_(H)H) domain derived from a camelid antibody.

In certain embodiments of Embodiment Set #1, the binding region maycomprise: (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of: amino acids 269to 387 of SEQ ID NOs: 26, 29, 30, or 36; amino acids 269 to 397 of SEQID NO:25; amino acids 381 to 500 of SEQ ID NO: 24 or 27; amino acids 401to 522 of SEQ ID NO:36, or amino acids 401 to 520 of SEQ ID NO:28; and(b) at least one light chain variable (V_(L)) domain comprising,consisting essentially of, or consisting of: amino acids 269 to 375 ofSEQ ID NO: 24, 27, or 28; amino acids 393 to 499 of SEQ ID NO:26; aminoacids 403 to 513 of SEQ ID NO:25; amino acids 408 to 514 of SEQ IDNO:36; and amino acids 413 to 519 of SEQ ID NO: 29 or 30. For example,the binding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:29; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 413 to 519 of SEQ ID NO:29. For example, thebinding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:36; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 408 to 514 of SEQ ID NO:36

In certain embodiments of Embodiment Set #1, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any one of the following polypeptide sequences: aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ IDNO:26; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; aminoacids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370 of SEQ IDNO:34; amino acids 253 to 367 of SEQ ID NO:35; or amino acids 269 to 514of SEQ ID NO:36. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 269 to 519 of SEQ ID NO:29. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 268 to 518 of SEQ ID NO: 102. For example,the binding region comprises, consists essentially of, or consists ofthe polypeptide represented by amino acids 268 to 386 of SEQ ID NO:31.For example, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 253 to 370 of SEQID NO:34. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 253 to 367 of SEQ ID NO:35. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 269 to 514 of SEQ ID NO:36.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention comprises, consists essentially of, or consistsof the polypeptide shown in any one of SEQ ID NOs: 22-36 and 97-108. Incertain embodiments of Embodiment Set #1, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofthe polypeptide shown in any one of SEQ ID NOs: 29, 31, 34, 35, 36, 102,104, and 106-108. In certain further embodiments, the cell-targetingmolecule of the present invention further comprises an amino terminalmethionine residue. In certain further embodiments, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of the polypeptide shown in SEQ ID NO: 29 or 102.

In certain embodiments of Embodiment Set #1, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set # 1, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #1, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set # 1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is capable when introduced to cells of exhibiting cytotoxicitythat is greater than that of a third cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the third cell-targeting molecule. Forcertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the third cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.In certain further embodiments, the third cell-targeting molecule doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family (e.g. SEQ ID NO:109).

In certain embodiments of Embodiment Set #1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a fifth cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the fifth cell-targeting molecule. In certain furtherembodiments, the fifth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Set # 1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention exhibits low cytotoxic potency (i.e. is not capable whenintroduced to certain positive target cell types of exhibiting acytotoxicity greater than 1% cell death of a cell population at acell-targeting molecule concentration of 1000 nM, 500 nM, 100 nM, 75 nM,or 50 nM) and is capable when introduced to cells of exhibiting agreater subcellular routing efficiency from an extracellular space to asubcellular compartment of an endoplasmic reticulum and/or cytosol ascompared to the cytotoxicity of a reference cell-targeting molecule,such as, e.g., a fifth cell-targeting molecule having an amino-terminusand comprising the binding region and the Shiga toxin effectorpolypeptide which is not positioned at or proximal to the amino-terminusof the fifth cell-targeting molecule. In certain further embodiments,the fifth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention, or a polypeptide component thereof, comprisesa carboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif of a member of the KDEL family. For certain further embodiments,the carboxy-terminal endoplasmic reticulum retention/retrieval signalmotif is selected from the group consisting of: KDEL (SEQ ID NO: 109),HDEF (SEQ ID NO: 110), HDEL (SEQ ID NO: 111), RDEF (SEQ ID NO: 112),RDEL (SEQ ID NO: 113), WDEL (SEQ ID NO: 114), YDEL (SEQ ID NO: 115),HEEF (SEQ ID NO: 116), HEEL (SEQ ID NO: 117), KEEL (SEQ ID NO: 118),REEL (SEQ ID NO: 119), KAEL (SEQ ID NO: 120), KCEL (SEQ ID NO: 121),KFEL (SEQ ID NO: 122), KGEL (SEQ ID NO: 123), KHEL (SEQ ID NO: 124),KLEL (SEQ ID NO: 125), KNEL (SEQ ID NO: 126), KQEL (SEQ ID NO: 127),KREL (SEQ ID NO: 128), KSEL (SEQ ID NO: 129), KVEL (SEQ ID NO: 130),KWEL (SEQ ID NO: 131), KYEL (SEQ ID NO: 132), KEDL (SEQ ID NO: 133),KIEL (SEQ ID NO: 134), DKEL (SEQ ID NO: 135), FDEL (SEQ ID NO: 136),KDEF (SEQ ID NO: 137), KKEL (SEQ ID NO: 138), HADL (SEQ ID NO: 139),HAEL (SEQ ID NO: 140), HIEL (SEQ ID NO: 141), HNEL (SEQ ID NO: 142),HTEL (SEQ ID NO: 143), KTEL (SEQ ID NO: 144), HVEL (SEQ ID NO: 145),NDEL (SEQ ID NO: 146), QDEL (SEQ ID NO: 147), REDL (SEQ ID NO: 148),RNEL (SEQ ID NO: 149), RTDL (SEQ ID NO: 150), RTEL (SEQ ID NO: 151),SDEL (SEQ ID NO: 152), TDEL (SEQ ID NO: 153), SKEL (SEQ ID NO: 154),STEL (SEQ ID NO: 155), and EDEL (SEQ ID NO: 156). In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable when introduced to cells of exhibiting cytotoxicity that isgreater than that of a reference molecule, such as, e.g., a sixthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the sixth cell-targeting molecule. Incertain further embodiments, the cell-targeting molecule of the presentinvention is capable of exhibiting a cytotoxicity with better optimized,cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, orgreater cytotoxicity as compared to the sixth cell-targeting molecule.In certain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the sixth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.

Embodiment Set #2 - HER2-Targeting Molecule Comprising a Shiga ToxinEffector Polypeptide Comprising (i) an Embedded or Inserted,Heterologous. T-Cell Epitope and (ii) a Disrupted, Furin-Cleavage Motif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule (HER2/neu/ErbB2) and (ii) a Shiga toxin A Subuniteffector polypeptide comprising an inserted or embedded, heterologous,epitope; and (iii) a disrupted furin-cleavage motif. In certainembodiments, the cell-targeting molecule of the present inventioncomprises (i) a binding region capable of specifically binding anextracellular target biomolecule; (ii) a Shiga toxin effectorpolypeptide comprising a Shiga toxin A1 fragment derived region and acarboxy terminus, wherein the Shiga toxin effector polypeptidecomprises: (a) an inserted or embedded, heterologous, epitope; and (b) adisrupted furin-cleavage motif at the carboxy-terminus of the A1fragment region. For certain further embodiments, the Shiga toxineffector polypeptide is capable of exhibiting at least one Shiga toxineffector function, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainembodiments, the furin-cleavage motif is disrupted by a carboxy-terminaltruncation of the Shiga toxin effector polypeptide as compared to thecarboxy-terminus of a wild-type Shiga toxin A Subunit. The Shiga toxineffector polypeptide may be truncated at its carboxy-terminus, relativeto a wild-type Shiga toxin A subunit, resulting in the elimination ofone or more endogenous, B-cell and/or CD4+ T-cell epitope regions. TheShiga toxin effector polypeptide of Embodiment Set #2 may furthercomprise at least one disrupted, endogenous, B-cell and/or CD4+ T-cellepitope region. In certain embodiments, the at least one disrupted,endogenous, B-cell and/or CD4+ T-cell epitope region does not overlapwith the embedded or inserted, heterologous, epitope. For example, thepresent invention provides a Shiga toxin effector polypeptide comprisinga Shiga toxin A1 fragment region and a carboxy-terminus, wherein theShiga toxin A subunit effector polypeptide comprises: (a) an embedded orinserted, heterologous, epitope; (b) a disruption of at least one,endogenous, B-cell and/or CD4+ T-cell epitope region; and (c) adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment region; wherein the Shiga toxin A subunit effectorpolypeptide is capable of exhibiting a Shiga toxin effector function. Ina further example, the present invention provides a Shiga toxin Asubunit effector polypeptide comprising a Shiga toxin A1 fragment regionand a carboxy-terminus, wherein the Shiga toxin A subunit effectorpolypeptide comprises (a) an embedded or inserted, heterologous, CD8+T-cell epitope which disrupts an endogenous, B-cell and/or CD4+ T-cellepitope region; (b) a disruption of at least four, endogenous, B-celland/or CD4+ T-cell epitope regions which do not overlap with theembedded or inserted, heterologous, CD8+ T-cell epitope; and (c) adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment region; wherein the Shiga toxin A subunit effectorpolypeptide is truncated at its carboxy-terminus, relative to awild-type Shiga toxin A subunit, resulting in the elimination of one ormore endogenous, B-cell and/or CD4+ T-cell epitope regions; and whereinthe Shiga toxin A subunit effector polypeptide is capable of exhibitinga Shiga toxin effector function. In certain further embodiments, theheterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g.,with regard to a human immune system. For certain further embodiments,the heterologous, T-cell epitope is capable of being presented by a MHCclass I molecule of a cell. In certain further embodiments, thecell-targeting molecule of the present invention is capable of one ormore the following: entering a cell, inhibiting a ribosome function,causing cytostasis, causing cell death, and/or delivering the embeddedor inserted, heterologous, T-cell epitope to a MHC class I molecule forpresentation on a cellular surface. For certain further embodiments, thecell-targeting molecule is capable when introduced to cells ofexhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a second cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide components comprise a wild-type Shiga toxin furin-cleavagesite at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #2, the binding region andShiga toxin effector polypeptide are linked together, either directly orindirectly.

In certain embodiments of Embodiment Set #2, the binding regioncomprises a polypeptide comprising an immunoglobulin orimmunoglobulin-type binding region. In certain further embodiments, thebinding region comprising a polypeptide selected from the groupconsisting of: an autonomous V_(H) domain, single-domain antibodyfragment (sdAb), nanobody®, heavy chain-antibody domain derived from acamelid antibody (V_(H)H or V_(H) domain fragment), heavy-chain antibodydomain derived from a cartilaginous fish antibody (V_(H)H or V_(H)domain fragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality. In certainembodiments, the binding region comprises a polypeptide selected fromthe group consisting of: an autonomous V_(H) domain, single-domainantibody fragment (sdAb), nanobody®, heavy chain-antibody domain derivedfrom a camelid antibody (V_(H)H or V_(H) domain fragment), heavy-chainantibody domain derived from a cartilaginous fish antibody (V_(H)H orV_(H) domain fragment), immunoglobulin new antigen receptor (IgNAR),V_(NAR) fragment, single-chain variable fragment (scFv), antibodyvariable fragment (Fv), Fd fragment, and antigen-binding fragment (Fab).In certain embodiments, the cell-targeting molecule of the presentinvention comprises an immunoglobulin binding region capable ofspecifically binding an extracellular part of HER2/neu/ErbB2, andcomprising one or more of: an antibody variable fragment, asingle-domain antibody fragment, a single-chain variable fragment, a Fdfragment, an antigen-binding fragment, an autonomous VH domain, a V_(H)Hfragment derived from a camelid antibody, a heavy-chain antibody domainderived from a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor. In certain embodiments, the bindingregion comprises, consists essentially of, or consists of a single-chainvariable fragment (scFv). In certain embodiments, the binding regioncomprises a single-chain variable fragment (scFv). In certainembodiments, the binding region comprises, consists essentially of, orconsists of a V_(H)H fragment derived from a camelid antibody.

In certain embodiments of Embodiment Set #2, the binding region and theShiga toxin effector polypeptide are fused, either directly orindirectly, forming a continuous polypeptide such that the bindingregion is associated, either directly or indirectly, with thecarboxy-terminus of the Shiga toxin effector polypeptide.

In certain embodiments of Embodiment Set #2, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/or CD4+T-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO: 1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQID NO:3; and 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or theequivalent region in a Shiga toxin A Subunit or derivative thereof; (ii)94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO: 1 orSEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ IDNO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 ofSEQ ID NO:3; 243-257 of SEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ IDNO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3;and 285-293 of SEQ ID NO:1 or SEQ ID NO:2, or the equivalent region in aShiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Set #2, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at (1) at 248-251 ofthe A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1), Shiga toxin (SEQ IDNO:2), or another Shiga toxin 1 variant sequence (e.g. SEQ ID NOs: 4-6);or (2) at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3)or a Shiga-like toxin 2 variant sequence (e.g. SEQ ID NOs: 7-18); or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #2, the Shiga toxin effectorpolypeptide comprises, consists essentially of, or consists of: (i)amino acids 75 to 251 of any one of SEQ ID NOs: 1-6 and 37; (ii) aminoacids 1 to 241 of any one of SEQ ID NOs: 1-18 and 75-89; (iii) aminoacids 1 to 251 of any one of SEQ ID NOs: 1-6, 37, and 75-89; or (iv)amino acids 1 to 261 of any one of SEQ ID NOs: 1-3; wherein the Shigatoxin effector polypeptide comprises at least one embedded or inserted,heterologous T-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #2, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #2, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #2, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #2, the binding region maycomprise at least one heavy-chain variable domain polypeptide comprising(i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ IDNOs: 51, SEQ ID NO:52, and SEQ ID NO:53, respectively; and at least onelight-chain variable domain polypeptide comprising: (i) the LCDR1,LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:54, SEQ IDNO:55, and SEQ ID NO:56, respectively. For example, the binding regionmay comprises at least one heavy-chain variable domain polypeptidecomprising (i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown inSEQ ID NOs: 57, SEQ ID NO:58, and SEQ ID NO:59, respectively; and atleast one light-chain variable domain polypeptide comprising (i) theLCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:60, SEQID NO:61, and SEQ ID NO:62, respectively. For example, the bindingregion may comprises at least one heavy-chain variable domainpolypeptide comprising (i) the HCDR1, HCDR2, and HCDR3 amino acidsequences shown in SEQ ID NOs: 63, SEQ ID NO:64, and SEQ ID NO:65,respectively; and at least one light-chain variable domain polypeptidecomprising (i) the LCDR1, LCDR2, and LCDR3 amino acid sequences shown inSEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively. The bindingregion having these CDRs may be an immunoglobulin binding regioncomprising a single-chain variable fragment.

In certain embodiments of Embodiment Set #2, the binding region maycomprises the binding region comprises a polypeptide selected from thegroup consisting of: a) a heavy chain only variable (V_(H)H) domaincomprising (i) a HCDR1 comprising or consisting essentially of the aminoacid sequences as shown in SEQ ID NO:69 or SEQ ID NO:72; (ii) a HCDR2comprising or consisting essentially of the amino acid sequence as shownin SEQ ID NO:70 or SEQ ID NO:73; and (iii) a HCDR3 comprising orconsisting essentially of the amino acid sequence as shown in SEQ IDNO:71 or SEQ ID NO:74. The binding region having these CDRs may be animmunoglobulin binding region comprising a heavy chain only variable(V_(H)H) domain derived from a camelid antibody.

In certain embodiments of Embodiment Set #2, the binding region maycomprise: (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of: amino acids 269to 387 of SEQ ID NOs: 26, 29, 30, or 36; amino acids 269 to 397 of SEQID NO:25; amino acids 381 to 500 of SEQ ID NO: 24 or 27; amino acids 401to 522 of SEQ ID NO:36, or amino acids 401 to 520 of SEQ ID NO:28; and(b) at least one light chain variable (V_(L)) domain comprising,consisting essentially of, or consisting of: amino acids 269 to 375 ofSEQ ID NO: 24, 27, or 28; amino acids 393 to 499 of SEQ ID NO:26; aminoacids 403 to 513 of SEQ ID NO:25; amino acids 408 to 514 of SEQ IDNO:36; and amino acids 413 to 519 of SEQ ID NO: 29 or 30. For example,the binding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:29; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 413 to 519 of SEQ ID NO:29. For example, thebinding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:36; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 408 to 514 of SEQ ID NO:36

In certain embodiments of Embodiment Set #2, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any one of the following polypeptide sequences: aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ IDNO:26; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; aminoacids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370 of SEQ IDNO:34; amino acids 253 to 367 of SEQ ID NO:35; or amino acids 269 to 514of SEQ ID NO:36. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 269 to 519 of SEQ ID NO:29. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 268 to 518 of SEQ ID NO: 102. For example,the binding region comprises, consists essentially of, or consists ofthe polypeptide represented by amino acids 268 to 386 of SEQ ID NO:31.For example, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 253 to 370 of SEQID NO:34. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 253 to 367 of SEQ ID NO:35. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 269 to 514 of SEQ ID NO:36.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention comprises, consists essentially of, or consistsof the polypeptide shown in any one of SEQ ID NOs: 22-36 and 97-108. Incertain embodiments of Embodiment Set #2, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofthe polypeptide shown in any one of SEQ ID NOs: 29, 31, 34, 35, 36, 102,104, and 106-108. In certain further embodiments, the cell-targetingmolecule of the present invention further comprises an amino terminalmethionine residue. In certain further embodiments, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of the polypeptide shown in SEQ ID NO: 29 or 102.

In certain embodiments of Embodiment Set #2, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a reference cell-targeting molecule,such as, e.g., a fourth cell-targeting molecule consisting of thecell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxyterminus of its A1 fragment region. In certain further embodiments, thecell-targeting molecule of the present invention is capable whenintroduced to cells of exhibiting cytotoxicity that is in a range offrom 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold,2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by thefourth cell-targeting molecule.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of a reference molecule,such as, e.g., a fourth cell-targeting molecule consisting of thecell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxyterminus of its A1 fragment region.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., a seventhcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference molecule, such as, e.g., fourth cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage siteat the carboxy terminus of its A1 fragment region.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule exhibits reduced relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., awild-type Shiga toxin A Subunit or a third cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment.

Embodiment Set #3 - HER2-Targeting Molecule Comprising a De-ImmunizedShiga Toxin Effector Polypeptide Comprising a Disrupted, Furin-CleavageMotif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule (HER2/neu/ErbB2) and (ii) a de-immunized, Shiga toxinA Subunit effector polypeptide comprising a disrupted furin-cleavagemotif. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule and (ii) a de-immunized,Shiga toxin effector polypeptide comprising a Shiga toxin A1 fragmentderived region and a carboxy terminus, wherein the Shiga toxin effectorpolypeptide comprises (a) a disrupted furin-cleavage motif at thecarboxy-terminus of the A1 fragment region, and (b) at least onedisrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitoperegion. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. In certain embodiments, the furin-cleavage motif is disrupted bya carboxy-terminal truncation of the Shiga toxin effector polypeptide ascompared to the carboxy-terminus of a wild-type Shiga toxin A Subunit.The Shiga toxin effector polypeptide may be truncated at itscarboxy-terminus, relative to a wild-type Shiga toxin A subunit,resulting in the elimination of one or more endogenous, B-cell and/orCD4+ T-cell epitope regions. The Shiga toxin effector polypeptide ofEmbodiment Set #3 may further comprise an inserted or embedded,heterologous, epitope; such as an embedded or inserted, heterologous,CD8+ T-cell epitope. The embedded or inserted, heterologous, CD8+ T-cellepitope may disrupt an endogenous, B-cell and/or CD4+ T-cell epitoperegion. For example, the present invention provides a Shiga toxineffector polypeptide comprising a Shiga toxin A1 fragment region and acarboxy-terminus, wherein the Shiga toxin A subunit effector polypeptidecomprises: a) an embedded or inserted, heterologous, epitope; (b) adisruption of at least one, endogenous, B-cell and/or CD4+ T-cellepitope region; and (c) a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment region; and wherein theShiga toxin A subunit effector polypeptide is capable of exhibiting aShiga toxin effector function. In a further example, the presentinvention provides a Shiga toxin A subunit effector polypeptidecomprising a Shiga toxin A1 fragment region and a carboxy-terminus,wherein the Shiga toxin A subunit effector polypeptide comprises: (a) anembedded or inserted, heterologous, CD8+ T-cell epitope which disruptsan endogenous, B-cell and/or CD4+ T-cell epitope region; (b) adisruption of at least four, endogenous, B-cell and/or CD4+ T-cellepitope regions which do not overlap with the embedded or inserted,heterologous, CD8+ T-cell epitope; and (c) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment region; andwherein the Shiga toxin A subunit effector polypeptide is truncated atits carboxy-terminus, relative to a wild-type Shiga toxin A subunit,resulting in the elimination of one or more endogenous, B-cell and/orCD4+ T-cell epitope regions; wherein the Shiga toxin A subunit effectorpolypeptide is capable of exhibiting a Shiga toxin effector function.

For certain embodiments of Embodiment Set #3, the cell-targetingmolecule exhibits reduced relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., awild-type Shiga toxin A Subunit or a third cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment.

In certain embodiments of Embodiment Set #3, the binding region andShiga toxin effector polypeptide are linked together, either directly orindirectly.

In certain embodiments of Embodiment Set #3, the binding regioncomprises a polypeptide comprising an immunoglobulin orimmunoglobulin-type binding region. In certain further embodiments, thebinding region comprising a polypeptide selected from the groupconsisting of: an autonomous V_(H) domain, single-domain antibodyfragment (sdAb), nanobody®, heavy chain-antibody domain derived from acamelid antibody (V_(H)H or V_(H) domain fragment), heavy-chain antibodydomain derived from a cartilaginous fish antibody (V_(H)H or V_(H)domain fragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality. In certainembodiments, the binding region comprises a polypeptide selected fromthe group consisting of: an autonomous V_(H) domain, single-domainantibody fragment (sdAb), nanobody®, heavy chain-antibody domain derivedfrom a camelid antibody (V_(H)H or V_(H) domain fragment), heavy-chainantibody domain derived from a cartilaginous fish antibody (V_(H)H orV_(H) domain fragment), immunoglobulin new antigen receptor (IgNAR),V_(NAR) fragment, single-chain variable fragment (scFv), antibodyvariable fragment (Fv), Fd fragment, and antigen-binding fragment (Fab).In certain embodiments, the cell-targeting molecule of the presentinvention comprises an immunoglobulin binding region capable ofspecifically binding an extracellular part of HER2/neu/ErbB2, andcomprising one or more of: an antibody variable fragment, asingle-domain antibody fragment, a single-chain variable fragment, a Fdfragment, an antigen-binding fragment, an autonomous VH domain, a V_(H)Hfragment derived from a camelid antibody, a heavy-chain antibody domainderived from a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor. In certain embodiments, the bindingregion comprises, consists essentially of, or consists of a single-chainvariable fragment (scFv). In certain embodiments, the binding regioncomprises a single-chain variable fragment (scFv). In certainembodiments, the binding region comprises, consists essentially of, orconsists of a V_(H)H fragment derived from a camelid antibody.

In certain embodiments of Embodiment Set #3, the binding region and theShiga toxin effector polypeptide are fused, either directly orindirectly, forming a continuous polypeptide such that the bindingregion is associated, either directly or indirectly, with thecarboxy-terminus of the Shiga toxin effector polypeptide.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity. For certain furtherembodiments, the cell-targeting molecule exhibits reduced relativeantigenicity and/or relative immunogenicity as compared to a referencemolecule, such as, e.g., a wild-type Shiga toxin A Subunit or a thirdcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment.

For certain further embodiments, the cell-targeting molecule is capablewhen introduced to cells of exhibiting a cytotoxicity comparable orbetter than a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide components comprise a wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #3, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or CD4+ T-cell epitope region selected fromthe group of natively positioned Shiga toxin A Subunit regionsconsisting of: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3;26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3;179-190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 ofSEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2, and 210-218 of SEQ IDNO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2;254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297of SEQ ID NO:3; 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; 4-33 of SEQ IDNO: 1 or SEQ ID NO:2; 34-78 of SEQ ID NO: 1 or SEQ ID NO:2; 77-103 ofSEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2;160-183 of SEQ ID NO: 1 or SEQ ID NO:2; 236-258 of SEQ ID NO: 1 or SEQID NO:2; and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalentregion in a Shiga toxin A Subunit or derivative thereof. In certainfurther embodiments, there is no disruption which is a carboxy-terminaltruncation of amino acid residues that overlap with part or all of atleast one disrupted, endogenous, B-cell and/or CD4+ T-cell epitopeand/or epitope region (which may also disrupt an additional, different,endogenous, B-cell and/or CD4+ T-cell epitope region(s)).

In certain embodiments of Embodiment Set #3, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at (1) at 248-251 ofthe A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1), Shiga toxin (SEQ IDNO:2), or another Shiga toxin 1 variant sequence (e.g. SEQ ID NOs: 4-6);or (2) at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3)or a Shiga-like toxin 2 variant sequence (e.g. SEQ ID NOs: 7-18); or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #3, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #3, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #3, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #3, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #3, the Shiga toxin effectorpolypeptide comprises, consists essentially of, or consists of: (i)amino acids 75 to 251 of any one of SEQ ID NOs: 1-6 and 37; (ii) aminoacids 1 to 241 of any one of SEQ ID NOs: 1-18 and 75-89; (iii) aminoacids 1 to 251 of any one of SEQ ID NOs: 1-6, 37, and 75-89; or (iv)amino acids 1 to 261 of any one of SEQ ID NOs: 1-3; wherein the Shigatoxin effector polypeptide comprises at least one at least one (two,three, four or more) disrupted, endogenous, B-cell and/or CD4+ T-cellepitope and/or epitope region(s) and a disrupted furin-cleavage motif atthe carboxy-terminus of a Shiga toxin A1 fragment derived region.

In certain embodiments of Embodiment Set #3, the binding region maycomprise at least one heavy-chain variable domain polypeptide comprising(i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ IDNOs: 51, SEQ ID NO:52, and SEQ ID NO:53, respectively; and at least onelight-chain variable domain polypeptide comprising: (i) the LCDR1,LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:54, SEQ IDNO:55, and SEQ ID NO:56, respectively. For example, the binding regionmay comprises at least one heavy-chain variable domain polypeptidecomprising (i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown inSEQ ID NOs: 57, SEQ ID NO:58, and SEQ ID NO:59, respectively; and atleast one light-chain variable domain polypeptide comprising (i) theLCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:60, SEQID NO:61, and SEQ ID NO:62, respectively. For example, the bindingregion may comprises at least one heavy-chain variable domainpolypeptide comprising (i) the HCDR1, HCDR2, and HCDR3 amino acidsequences shown in SEQ ID NOs: 63, SEQ ID NO:64, and SEQ ID NO:65,respectively; and at least one light-chain variable domain polypeptidecomprising (i) the LCDR1, LCDR2, and LCDR3 amino acid sequences shown inSEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively. The bindingregion having these CDRs may be an immunoglobulin binding regioncomprising a single-chain variable fragment.

In certain embodiments of Embodiment Set #3, the binding region maycomprises the binding region comprises a polypeptide selected from thegroup consisting of: a) a heavy chain only variable (V_(H)H) domaincomprising (i) a HCDR1 comprising or consisting essentially of the aminoacid sequences as shown in SEQ ID NO:69 or SEQ ID NO:72; (ii) a HCDR2comprising or consisting essentially of the amino acid sequence as shownin SEQ ID NO:70 or SEQ ID NO:73; and (iii) a HCDR3 comprising orconsisting essentially of the amino acid sequence as shown in SEQ IDNO:71 or SEQ ID NO:74. The binding region having these CDRs may be animmunoglobulin binding region comprising a heavy chain only variable(V_(H)H) domain derived from a camelid antibody.

In certain embodiments of Embodiment Set #3, the binding region maycomprise: (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of: amino acids 269to 387 of SEQ ID NOs: 26, 29, 30, or 36; amino acids 269 to 397 of SEQID NO:25; amino acids 381 to 500 of SEQ ID NO: 24 or 27; amino acids 401to 522 of SEQ ID NO:36, or amino acids 401 to 520 of SEQ ID NO:28; and(b) at least one light chain variable (V_(L)) domain comprising,consisting essentially of, or consisting of: amino acids 269 to 375 ofSEQ ID NO: 24, 27, or 28; amino acids 393 to 499 of SEQ ID NO:26; aminoacids 403 to 513 of SEQ ID NO:25; amino acids 408 to 514 of SEQ IDNO:36; and amino acids 413 to 519 of SEQ ID NO: 29 or 30. For example,the binding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:29; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 413 to 519 of SEQ ID NO:29. For example, thebinding region may comprise (a) at least one heavy chain variable(V_(H)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 387 of SEQ ID NO:36; and (b) at least one light chainvariable (V_(L)) domain comprising, consisting essentially of, orconsisting of amino acids 408 to 514 of SEQ ID NO:36

In certain embodiments of Embodiment Set #3, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any one of the following polypeptide sequences: aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ IDNO:26; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; aminoacids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370 of SEQ IDNO:34; amino acids 253 to 367 of SEQ ID NO:35; or amino acids 269 to 514of SEQ ID NO:36. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 269 to 519 of SEQ ID NO:29. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 268 to 518 of SEQ ID NO: 102. For example,the binding region comprises, consists essentially of, or consists ofthe polypeptide represented by amino acids 268 to 386 of SEQ ID NO:31.For example, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 253 to 370 of SEQID NO:34. For example, the binding region comprises, consistsessentially of, or consists of the polypeptide represented by aminoacids 253 to 367 of SEQ ID NO:35. For example, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by amino acids 269 to 514 of SEQ ID NO:36.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeof the present invention comprises, consists essentially of, or consistsof the polypeptide shown in any one of SEQ ID NOs: 22-36 and 97-108. Incertain embodiments of Embodiment Set #3, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofthe polypeptide shown in any one of SEQ ID NOs: 29, 31, 34, 35, 36, 102,104, and 106-108. In certain further embodiments, the cell-targetingmolecule of the present invention further comprises an amino terminalmethionine residue. In certain further embodiments, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of the polypeptide shown in SEQ ID NO: 29 or 102.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a reference molecule, such as, e.g., afourth cell-targeting molecule consisting of the cell-targeting moleculeexcept for all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion. In certain further embodiments, the cell-targeting molecule ofthe present invention is capable when introduced to cells of exhibitingcytotoxicity that is in a range of from 0.1-fold, 0.5-fold, or 0.75-foldto 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold ofthe cytotoxicity exhibited by the fourth cell-targeting molecule.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of a reference molecule,such as, e.g., a fourth cell-targeting molecule consisting of thecell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxyterminus of its A1 fragment region.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule, such as, e.g., a fourthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

Further Embodiments of Embodiment Sets # 1-#3

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide is truncated at its carboxy-terminus, relative to awild-type Shiga toxin A subunit, resulting in the elimination of one ormore endogenous, B-cell and/or CD4+ T-cell epitope regions.

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide has a Shiga toxin A1 fragment derived region havinga carboxy terminus and further comprises a disrupted furin-cleavagemotif at the carboxy-terminus of the A1 fragment region. In certainembodiments, the furin-cleavage motif is disrupted by a carboxy-terminaltruncation of the Shiga toxin effector polypeptide as compared to thecarboxy-terminus of a wild-type Shiga toxin A Subunit. For example, theShiga toxin effector polypeptide of the present invention may comprise aShiga toxin A1 fragment derived region wherein the Shiga toxin A1fragment region comprises a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment region, and wherein saidfurin-cleavage motif is disrupted by a carboxy-terminal truncation ofthe Shiga toxin effector polypeptide as compared to the carboxy-terminusof a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide further comprises at least one inserted orembedded, heterologous epitope. In certain embodiments of EmbodimentSets # 1 to #3, the Shiga toxin effector polypeptide comprises at leastone embedded, heterologous epitope. In certain embodiments, the at leastone inserted or embedded, heterologous epitope is a CD8+ T-cell epitope.In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises at least one inserted or embedded,heterologous CD8+ T-cell epitope. In certain embodiments, the embeddedor inserted, heterologous, CD8+ T-cell epitope disrupts an endogenous,B-cell and/or CD4+ T-cell epitope region.

In certain embodiments of Embodiment Sets # 1 to #3, the amino-terminusof the Shiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., an fifth cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the fifth cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the fifth cell-targeting molecule. Forcertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.In certain further embodiments, the fifth cell-targeting molecule doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention, or a polypeptide component thereof,comprises a carboxy-terminal, endoplasmic reticulum retention/retrievalsignal motif of a member of the KDEL family. In certain furtherembodiments, the carboxy-terminal endoplasmic reticulumretention/retrieval signal motif is selected from the group consistingof: KDEL (SEQ ID NO: 109), HDEF (SEQ ID NO: 110), HDEL (SEQ ID NO: 111),RDEF (SEQ ID NO: 112), RDEL (SEQ ID NO: 113), WDEL (SEQ ID NO: 114),YDEL (SEQ ID NO: 115), HEEF (SEQ ID NO: 116), HEEL (SEQ ID NO: 117),KEEL (SEQ ID NO: 118), REEL (SEQ ID NO: 119), KAEL (SEQ ID NO: 120),KCEL (SEQ ID NO: 121), KFEL (SEQ ID NO: 122), KGEL (SEQ ID NO: 123),KHEL (SEQ ID NO: 124), KLEL (SEQ ID NO: 125), KNEL (SEQ ID NO: 126),KQEL (SEQ ID NO: 127), KREL (SEQ ID NO: 128), KSEL (SEQ ID NO: 129),KVEL (SEQ ID NO: 130), KWEL (SEQ ID NO: 131), KYEL (SEQ ID NO: 132),KEDL (SEQ ID NO: 133), KIEL (SEQ ID NO: 134), DKEL (SEQ ID NO: 135),FDEL (SEQ ID NO: 136), KDEF (SEQ ID NO: 137), KKEL (SEQ ID NO: 138),HADL (SEQ ID NO: 139), HAEL (SEQ ID NO: 140), HIEL (SEQ ID NO: 141),HNEL (SEQ ID NO: 142), HTEL (SEQ ID NO: 143), KTEL (SEQ ID NO: 144),HVEL (SEQ ID NO: 145), NDEL (SEQ ID NO: 146), QDEL (SEQ ID NO: 147),REDL (SEQ ID NO: 148), RNEL (SEQ ID NO: 149), RTDL (SEQ ID NO: 150),RTEL (SEQ ID NO: 151), SDEL (SEQ ID NO: 152), TDEL (SEQ ID NO: 153),SKEL (SEQ ID NO: 154), STEL (SEQ ID NO: 155), and EDEL (SEQ ID NO: 156).In certain further embodiments, the cell-targeting molecule of thepresent invention is capable when introduced to cells of exhibitingcytotoxicity that is greater than that of a reference molecule, such as,e.g., a sixth cell-targeting molecule consisting of the cell-targetingmolecule except for it does not comprise any carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif of the KDELfamily. In certain further embodiments, the cell-targeting molecule ofthe present invention is capable of exhibiting a cytotoxicity withbetter optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold,6-fold, 9-fold, or greater cytotoxicity as compared to a referencemolecule, such as, e.g., a sixth cell-targeting molecule consisting ofthe cell-targeting molecule except for it does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cytotoxicity ofthe cell-targeting molecule of the present invention to a population oftarget positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold or greater than the cytotoxicity of the sixthcell-targeting molecule to a second population of target positive cellsas assayed by CD₅₀ values.

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide further comprises at least one, two, three, four,five, six, seven, or eight disrupted, endogenous, B-cell and/or T-cellepitope regions. In certain further embodiments, the Shiga toxineffector polypeptide comprises a disruption of at least one, two, three,four, five, six, seven, or eight endogenous, B-cell and/or CD4+ T-cellepitopes and/or epitope regions described herein. In certain furtherembodiments, the Shiga toxin effector polypeptide further comprises atleast one (such as at least two, three, four, five, six, seven, oreight) disrupted, endogenous, B-cell and/or CD4+ T-cell epitope regionwhich does not overlap with at least one inserted or embedded,heterologous epitope, which may also disrupt an additional, different,endogenous, B-cell and/or CD4+ T-cell epitope region(s). In certainembodiments, the Shiga toxin effector polypeptide comprises a disruptionof at least three, endogenous, B-cell and/or CD4+ T-cell epitope regionswhich do not overlap with the embedded or inserted, heterologous, CD8+T-cell epitope, which may also disrupt an additional, different,endogenous, B-cell and/or CD4+ T-cell epitope region(s).

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide further comprises a disruption in the endogenous,B-cell and/or CD4+ T-cell epitope region selected from the group ofnatively positioned Shiga toxin A Subunit regions consisting of: 1-15 ofSEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3;27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ IDNO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 ofSEQ ID NO: 1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ IDNO: 1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 197 of SEQ ID NO:3; 198 ofSEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO: 3; 205 of SEQ ID NO: 1 orSEQ ID NO: 2, and 210-218 of SEQ ID NO: 3; 240-260 of SEQ ID NO: 3;243-257 of SEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; 285-293 of SEQID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO: 1 or SEQ ID NO:2; 34-78 ofSEQ ID NO: 1 or SEQ ID NO:2; 77-103 of SEQ ID NO: 1 or SEQ ID NO:2;128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ ID NO: 1 or SEQID NO:2; 236-258 of SEQ ID NO: 1 or SEQ ID NO:2; and 274-293 of SEQ IDNO: 1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin ASubunit or derivative thereof. In certain further embodiments, there isno disruption which is a carboxy-terminal truncation of amino acidresidues that overlap with part or all of at least one disrupted,endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region.

In certain embodiments of Embodiment Sets # 1 to #3, the Shiga toxineffector polypeptide further comprises a disruption of at least one(such as at least two, three, four, five, six, seven, eight or more)endogenous, B-cell and/or CD4+ T-cell epitope region, wherein the B-cellregion is selected from the group of natively positioned Shiga toxin ASubunit regions consisting of: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ IDNO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2;140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2;197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 210-218 of SEQ IDNO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: 1 or SEQ ID NO:2;254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in the Shiga toxin effector polypeptides SEQ IDNOs: 4-18); and the CD4+ T-cell epitope region is selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 4-33 of SEQ ID NO: 1 or SEQ ID NO:2; 34-78 of SEQ ID NO: 1 or SEQ IDNO:2; 77-103 of SEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 orSEQ ID NO:2; 160-183 of SEQ ID NO: 1 or SEQ ID NO:2; 236-258 of SEQ IDNO: 1 or SEQ ID NO:2; and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in the Shiga toxin effector polypeptides SEQ IDNOs: 4-18). In certain embodiments, the B-cell epitope region isselected from the group of natively positioned Shiga toxin A Subunitregions consisting of: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2;39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2; 140-156 ofSEQ ID NO:3; 179-190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191 of SEQ IDNO:3; 197 of SEQ ID NO:3; 198 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 210-218 of SEQ ID NO:3; and243-257 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in aShiga toxin A Subunit or derivative thereof (such as the equivalentregion in the Shiga toxin effector polypeptides SEQ ID NOs: 4-18); andthe CD4+ T-cell epitope region is selected from the group of nativelypositioned Shiga toxin A Subunit regions consisting of: 4-33 of SEQ IDNO: 1 or SEQ ID NO:2; 34-78 of SEQ ID NO: 1 or SEQ ID NO:2; 77-103 ofSEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2;160-183 of SEQ ID NO: 1 or SEQ ID NO:2; and 236-258 of SEQ ID NO: 1 orSEQ ID NO:2; or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in the Shiga toxineffector polypeptides SEQ ID NOs: 4-18). For example, the B-cell epitoperegion may be selected from the group of natively positioned Shiga toxinA Subunit regions consisting of: 39-48 of SEQ ID NO: 1 or SEQ ID NO:2;42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 179-190 ofSEQ ID NO:1 or SEQ ID NO:2; and 179-191 of SEQ ID NO:3; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in the Shiga toxin effector polypeptides SEQ IDNOs: 4-18); and the CD4+ T-cell epitope region is 236-258 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in the Shiga toxineffector polypeptides SEQ ID NOs: 4-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises a disruption of at least four,endogenous, B-cell and/or CD4+ T-cell epitope regions, wherein thedisruption comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell epitope region selected from the group ofnatively positioned Shiga toxin A Subunit regions consisting of: 1-15 ofSEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3;27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ IDNO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 ofSEQ ID NO: 1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ IDNO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 197 of SEQ ID NO:3; 198 ofSEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 orSEQ ID NO:2; 210-218 of SEQ ID NO:3; and 243-257 of SEQ ID NO: 1 or SEQID NO:2; or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in the Shiga toxineffector polypeptides SEQ ID NOs: 4-18); and/or the CD4+ T-cell epitoperegion selected from the group of natively positioned Shiga toxin ASubunit regions consisting of: 4-33 of SEQ ID NO: 1 or SEQ ID NO:2;34-78 of SEQ ID NO: 1 or SEQ ID NO:2; 77-103 of SEQ ID NO: 1 or SEQ IDNO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ ID NO: 1 orSEQ ID NO:2; and 236-258 of SEQ ID NO: 1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in the Shiga toxin effector polypeptides SEQ IDNOs: 4-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide further comprises a mutation, relative to awild-type Shiga toxin A Subunit, in the B-cell immunogenic amino acidresidue selected from the group of natively positioned Shiga toxin ASubunit amino acid residues: L49, D197, D198, R204, and R205.

In certain embodiments of Embodiment Sets #1 to #3, the embedded orinserted, heterologous, T-cell epitope disrupts the endogenous, B-celland/or CD4+ T-cell epitope region is selected from the group of nativelypositioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of SEQID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3;27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ IDNO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in any one of theShiga toxin 1 effector polypeptide variants shown in SEQ ID NOs: 4-6 andany one of the Shiga-like toxin 2 effector polypeptide variants shown inSEQ ID NOs: 7-18), wherein there is no disruption which is anamino-terminal truncation of sequences that overlap with part or all ofat least one disrupted epitope region; (ii) 94-115 of SEQ ID NO: 1, SEQID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 or SEQ ID NO:2; 140-156of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ IDNO:3; 197 of SEQ ID NO:3; 198 of SEQ ID NO: 1 or SEQ ID NO:2; 204 of SEQID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3;and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: 1 or SEQ IDNO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2; orthe equivalent region in a Shiga toxin A Subunit or derivative thereof(such as the equivalent region in any one of the Shiga toxin 1 effectorpolypeptide variants shown in SEQ ID NOs: 4-6 and any one of theShiga-like toxin 2 effector polypeptide variants shown in SEQ ID NOs:7-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises a mutation, relative to a wild-type Shigatoxin A Subunit, in the B-cell and/or CD4+ T-cell epitope regionselected from the group of natively positioned Shiga toxin A Subunitregions consisting of: (i) 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 ofSEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2;39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; or the equivalent region in aShiga toxin A Subunit or derivative thereof (such as the equivalentregion in any one of the Shiga toxin 1 effector polypeptide variantsshown in SEQ ID NOs: 4-6 and any one of the Shiga-like toxin 2 effectorpolypeptide variants shown in SEQ ID NOs: 7-18), wherein there is nodisruption which is an amino-terminal truncation of sequences thatoverlap with part or all of at least one disrupted epitope region; (ii)94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO: 1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO: 1 orSEQ ID NO:2; 179-191 of SEQ ID NO:3; 197 of SEQ ID NO:3; 198 of SEQ IDNO: 1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ IDNO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3;243-257 of SEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 ofSEQ ID NO: 1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin ASubunit or derivative thereof (such as the equivalent region in any oneof the Shiga toxin 1 effector polypeptide variants shown in SEQ ID NOs:4-6 and any one of the Shiga-like toxin 2 effector polypeptide variantsshown in SEQ ID NOs: 7-18), wherein there is no disruption which is anamino-terminal truncation of sequences that overlap with part or all ofat least one disrupted epitope region.

In certain embodiments of Embodiment Sets #1 to #3, the embedded orinserted, heterologous, CD8+ T-cell epitope disrupts an endogenous,B-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: 1-15 of SEQ ID NO: 1 or SEQID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ IDNO:3; and 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, 94-115 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO: 1 orSEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ IDNO:2; 179-191 of SEQ ID NO:3; 197 of SEQ ID NO:3; 198 of SEQ ID NO: 1 orSEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ IDNO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2,or the equivalent region in a Shiga toxin A Subunit or derivativethereof (such as the equivalent region in any one of the Shiga toxin 1effector polypeptide variants shown in SEQ ID NOs: 4-6 and any one ofthe Shiga-like toxin 2 effector polypeptide variants shown in SEQ IDNOs: 7-18); and/or an endogenous CD4+ T-cell epitope region selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: 4-33 of SEQ ID NO:1 or SEQ ID NO:2; 34-78 of SEQ ID NO:1or SEQ ID NO:2; 77-103 of SEQ ID NO:1 or SEQ ID NO:2; 128-168 of SEQ IDNO:1 or SEQ ID NO:2; 160-183 of SEQ ID NO:1 or SEQ ID NO:2; 236-258 ofSEQ ID NO:1 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQ ID NO:2 orthe equivalent region in a Shiga toxin A Subunit or derivative thereof(such as the equivalent region in any one of the Shiga toxin 1 effectorpolypeptide variants shown in SEQ ID NOs: 4-6 and any one of theShiga-like toxin 2 effector polypeptide variants shown in SEQ ID NOs:7-18).

In certain embodiments of Embodiment Sets #1 to #3, the embedded orinserted, heterologous, CD8+ T-cell epitope disrupts an endogenous,B-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: 1-15 of SEQ ID NO:1 or SEQID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ IDNO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ IDNO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218of SEQ ID NO:3; 240-260 of SEQ ID NO:3; and 243-257 of SEQ ID NO:1 orSEQ ID NO:2, or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in any one of theShiga toxin 1 effector polypeptide variants shown in SEQ ID NOs: 4-6 andany one of the Shiga-like toxin 2 effector polypeptide variants shown inSEQ ID NOs: 7-18); and/or an endogenous CD4+ T-cell epitope regionselected from the group of natively positioned Shiga toxin A Subunitregions consisting of: 4-33 of SEQ ID NO:1 or SEQ ID NO:2; 34-78 of SEQID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 or SEQ ID NO:2; 128-168 ofSEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ ID NO:1 or SEQ ID NO:2; and236-258 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent region in aShiga toxin A Subunit or derivative thereof (such as the equivalentregion in any one of the Shiga toxin 1 effector polypeptide variantsshown in SEQ ID NOs: 4-6 and any one of the Shiga-like toxin 2 effectorpolypeptide variants shown in SEQ ID NOs: 7-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises a disruption of at least one endogenousepitope region selected from the group of natively positioned Shigatoxin A Subunits consisting of: 94-115 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ IDNO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 197of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3;205 of SEQ ID NO:1 or SEQ ID NO:2; or 210-218 of SEQ ID NO:3 or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas in any one of the Shiga toxin 1 effector polypeptide variants shownin SEQ ID NOs: 4-6 and in any one of the Shiga-like toxin 2 effectorpolypeptide variants shown in SEQ ID NOs: 7-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide does not comprise a heterologous, MHC classI-restricted, T-cell epitope. MHC class I-restricted, T-cell epitopesare known in the art or can be predicted by the skilled worker. The termheterologous refers to MHC class I-restricted, T-cell epitopes which arenot natively present in wild-type Shiga toxin A Subunits, such as, e.g.,the wild-type Shiga toxin A Subunit which is most closely related to theShiga toxin effector polypeptide of interest.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises disruptions of at least two, three, four,five, six, seven, eight or more endogenous, B-cell and/or T-cell epitoperegions. In certain embodiments of Embodiment Sets #1 to #3, the Shigatoxin effector polypeptide comprises disruptions of at least fourendogenous, B-cell and/or T-cell epitope regions. In certainembodiments, the Shiga toxin effector polypeptide comprises disruptionsof at least five endogenous, B-cell and/or CD4+ T-cell epitope regions.For example in certain embodiments, the Shiga toxin effector polypeptidecomprises disruptions of at least six endogenous, B-cell and/or CD4+T-cell epitope regions. In certain further embodiments, the two, three,four, five, six, seven, eight or more disrupted epitope regions do notoverlap with the embedded or inserted, heterologous, CD8+ T-cellepitope, which may also disrupt an additional, different, endogenous,B-cell and/or CD4+ T-cell epitope region(s).

In certain embodiments of Embodiment Sets #1 to #3, one or moredisruptions comprises an amino acid residue substitution relative to awild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #3, one or moreendogenous, B-cell and/or T-cell epitope regions comprises a pluralityof amino acid residue substitutions relative to a wild-type Shiga toxinA Subunit. In certain embodiments, at least three, four, five or more ofthe B-cell and/or CD4+ T-cell epitope region disruptions comprise anamino acid residue substitution relative to a wild-type Shiga toxin ASubunit.

In certain embodiments of Embodiment Sets #1 to #3, at least one, two,three, or four disruptions comprise a plurality of amino acid residuesubstitutions in the endogenous, B-cell and/or T-cell epitope regionrelative to a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #3, at least onedisruption comprises at least one, two, three, four, five, six, seven,eight or more amino acid residue substitutions relative to a wild-typeShiga toxin A Subunit, and optionally wherein at least one substitutionoccurs at the natively positioned Shiga toxin A Subunit amino acidresidue selected from the group consisting of: 1 of SEQ ID NO:1 or SEQID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ IDNO:2; 46 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO:1or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 50 of SEQ ID NO:1 or SEQ IDNO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ IDNO:2; 54 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO:1or SEQ ID NO:2; 56 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 57 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 ofSEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 or SEQ ID NO:2;109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 orSEQ ID NO:2; 111 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 112 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 ofSEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ IDNO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2;186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 orSEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 orSEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 242 of SEQ ID NO: 1or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3;248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ IDNO:1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ IDNO:2; or the equivalent amino acid residue in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in any one of theShiga toxin 1 effector polypeptide variants shown in SEQ ID NOs: 4-6 andin any one of the Shiga-like toxin 2 effector polypeptide variants shownin SEQ ID NOs: 7-18).

In certain embodiments of Embodiment Sets #1 to #3, at least onedisruption comprises at least one, two, three, four, five, six, seven,eight, or more amino acid residue substitutions relative to a wild-typeShiga toxin A Subunit, and optionally wherein at least one substitution(such as at least two, three, four, five, six, seven, eight or moreamino acid residue substitutions) occurs at the natively positionedShiga toxin A Subunit amino acid residue selected from the groupconsisting of: 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 12of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ IDNO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ IDNO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 58 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ IDNO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1or SEQ ID NO:2; 88 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ IDNO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 141 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2;179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 orSEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ IDNO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ IDNO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQID NO:1 or SEQ ID NO:2; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ IDNO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ IDNO:2; 250 of SEQ ID NO:3; and 251 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent amino acid residue in a Shiga toxin A Subunit or derivativethereof (such as the equivalent region in any one of the Shiga toxin 1effector polypeptide variants shown in SEQ ID NOs: 4-6 and any one ofthe Shiga-like toxin 2 effector polypeptide variants shown in SEQ IDNOs: 7-18). In certain embodiments, the at least one substitution occursat the natively positioned Shiga toxin A Subunit amino acid residues: 45of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1, SEQ ID NO:2; 55 of SEQID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2; 59 of SEQ IDNO:1, SEQ ID NO:2; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1or SEQ ID NO:2; 110 of SEQ ID NO:1 or SEQ ID NO:2; 141 of SEQ ID NO:1 orSEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 242 of SEQ ID NO:1 orSEQ ID NO:2; 248 of SEQ ID NO:1 or SEQ ID NO:2; and 251 of SEQ ID NO:1or SEQ ID NO:2.

In certain further embodiments, at least two disruptions each compriseat least one amino acid residue substitutions relative to a wild-typeShiga toxin A Subunit selected form the group consisting of: 1 of SEQ IDNO:1 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 ofSEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 53of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 58 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ IDNO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 94 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 179 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID SEQ ID NO:2, or SEQID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ IDNO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2;189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ IDNO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 250 of SEQID NO:3; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ IDNO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent amino acid residue in a Shiga toxin A Subunit or derivativethereof (such as the equivalent region in any one of the Shiga toxin 1effector polypeptide variants shown in SEQ ID NOs: 4-6 and any one ofthe Shiga-like toxin 2 effector polypeptide variants shown in SEQ IDNOs: 7-18).

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises disruption of at least three, endogenous,B-cell and/or CD4+ T-cell epitope regions selected from the group ofconsisting of: (i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ IDNO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3, or the equivalent region in a Shigatoxin A Subunit or derivative thereof (such as the equivalent region inany one of the Shiga toxin 1 effector polypeptide variants shown in SEQID NOs: 4-6 and any one of the Shiga-like toxin 2 effector polypeptidevariants shown in SEQ ID NOs: 7-18), wherein there is no disruptionwhich is an amino-terminal truncation of amino acid residues thatoverlap with part or all of at least one disrupted, endogenous, B-celland/or CD4+ T-cell epitope region; (ii) 94-115 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 ofSEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ IDNO:3; 197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3;and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2;254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in any one of the Shiga toxin 1 effectorpolypeptide variants shown in SEQ ID NOs: 4-6 and any one of theShiga-like toxin 2 effector polypeptide variants shown in SEQ ID NOs:7-18), wherein there is no disruption which is a carboxy-terminaltruncation of amino acid residues that overlap with part or all of atleast one disrupted, endogenous, B-cell and/or CD4+ T-cell epitopeand/or epitope region.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises disruptions of at least two, endogenous,B-cell and/or CD4+ T-cell epitope regions, wherein each disruptioncomprises one or more amino acid residue substitutions, and wherein theendogenous, B-cell and/or CD4+ T-cell epitope regions are selected fromthe group of natively positioned Shiga toxin A Subunit regionsconsisting of: 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 ofSEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in any one of the Shiga toxin 1 effectorpolypeptide variants shown in SEQ ID NOs: 4-6 and any one of theShiga-like toxin 2 effector polypeptide variants shown in SEQ ID NOs:7-18).

In certain embodiments of Embodiment Sets #1 to #3, the embedded orinserted, heterologous, T-cell epitope does not disrupt any endogenous,B-cell and/or CD4+ T-cell epitope region described herein.

In certain embodiments of Embodiment Sets #1 to #3, at least onedisruption comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, D to M, D to R, E to A, E to G, E to V, E to L, E to I, E to F, Eto S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, Fto L, F to I, G to A, G to P, H to A, H to G, H to V, H to L, H to I, Hto F, H to M, I to A, I to V, I to G, Ito C, Kto A, Kto G, Kto V, Kto L,Kto I, Kto M, Kto H, L to A, Lto V, L to G, Lto C, Nto A, N to G, N toV, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R toV, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S toA, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T toV, T to L, T to I, T to F, T to M, T to S, V to A, V to G, Y to A, Y toG, Y to V, Y to L, Y to I, Y to F, Y to M, and Y to T. In certainfurther embodiments, the one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, Eto N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, Hto I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, Kto H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, Pto A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, Rto M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, Sto I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, Tto M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y toM.

In certain embodiments of Embodiment Sets #1 to #3, at least one of thedisruption(s) comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit selected from the groupconsisting of: K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F,M, and S; D6 to A, G, V, L, I, F, S, Q and R; T8 to A, G, V, I, L, F,and M; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, and S;S9 to A, G, V, L, I, F, and M; K11 to A, G, V, L, I, F, M and H; T12 toA, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A,G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to Aand P; D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, M and F;L49 to A, V, C, and G; Y49 to A, G, V, L, I, F, M, and T; F50 to A, G,V, L, I, and T; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, I, andL; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; I57 to A,G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, F, S,and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M, T,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; R84 toA, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; I88 to A, V, C, andG; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, L, I, F, M; and N; A105 to L; T107 to A, G, V, L, I, F,M, and P; S107 to A, G, V, L, I, F, M, and P; L108 to A, V, C, and G;S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S;G110 to A; S112 to A, G, V, L, I, F, and M; D111 to A, G, V, L, I, F, S,Q, and T; S112 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S,and Q; G147 to A; V154 to A and G; R179 to A, G, V, L, I, F, M, Q, S, K,and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M,and S; D183 to A, G, V, L, I, F, S, and Q; D184 to A, G, V, L, I, F, S,and Q; L185 to A, G, V and C; S186 to A, G, V, I, L, F, and M; G187 toA; R188 to A, G, V, L, I, F, M, Q, S, K, and H; S189 to A, G, V, I, L,F, and M; D197 to A, G, V, L, I, F, S, and Q; D198 to A, G, V, L, I, F,S, and Q; R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V,L, I, F, M, Q, S, K and H; C242 to A, G and V; S247 to A, G, V, I, L, F,and M; Y247 to A, G, V, L, I, F, and M; R247 to A, G, V, L, I, F, M, Q,S, K, and H; R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G,V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, andH; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V,L, I, F, M, and S. In certain embodiments of Embodiment Sets #1 to #3,the one or more substitutions are selected from the group ofsubstitutions at native positions in a Shiga toxin A Subunit consistingof: K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I,S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F,L49A, F50T, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F,I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L,G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V,G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A,D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T,R188A, R188L, S189A, D197A, D198A, R204A, R205A, C242A, S247I, Y247A,R247A, R248A, R250A, R251A, D264A, G264A, T286A, and T286I. In certainembodiments, the one or more substitutions are selected from the groupof substitutions at native positions in a Shiga toxin A Subunitconsisting of: K1A, S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A,D141A, G147A, R188A, C242S, R248A, and R251A. In certain furtherembodiments, the Shiga toxin effector polypeptide comprises one or moresubstitutions selected from the group of substitutions at nativepositions in a Shiga toxin A Subunit consisting of: K1R and K11R. Incertain further embodiments, the Shiga toxin effector polypeptidecomprises all the following substitutions: S45I, V54I, R55L, I57F, P59F,E60T, E61L, G110A, R188A, C242S, R248A, and R251A. In certain otherfurther embodiments, the Shiga toxin effector polypeptide comprises allthe following substitutions: K1A, S45I, V54I, R55L, I57F, P59F, E60T,E61L, G110A, G147A, C242S, R248A, and R251A. In certain other furtherembodiments, the Shiga toxin effector polypeptide comprises all thefollowing substitutions: S45I, V54I, R55L, I57F, P59F, E60T, E61L,G110A, D141A, R188A, C242S, R248A, and R251A. In certain furtherembodiments, the Shiga toxin effector polypeptide comprises all thefollowing substitutions: K1R, K11R, S45I, V54I, R55L, I57F, P59F, E60T,E61L, G110A, D141A, R188A, C242S, R248A, and R251A. In certainembodiments, the Shiga toxin effector polypeptide further comprises oneor more additional substitutions selected from the group ofsubstitutions at native positions in a Shiga toxin A Subunit consistingof: K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I,S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F,L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P,I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V,E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V,T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I,D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A,G187T, R188A, R188L, S189A, D197A, D198A, R204A, R205A, C242S, S247I,R247A, Y247A, R248A, R250A, and R251A.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention comprises the Shiga toxin effectorpolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an aminoacid sequence selected from any one of SEQ ID NOs: 19-21 and 75-89. Incertain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention comprises the Shiga toxin effectorpolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an aminoacid sequence selected from any one of SEQ ID NOs: 19-21. In certainembodiments of Embodiment Sets #1 to #3, the cell-targeting molecule ofthe present invention comprises the Shiga toxin effector polypeptidecomprising, consisting essentially of, or consisting of the polypeptideshown in any one of SEQ ID NOs: 19-21 and 75-89. In certain embodiments,the cell-targeting molecule of the present invention comprises the Shigatoxin effector polypeptide comprising, consisting essentially of, orconsisting of the polypeptide shown in any one of SEQ ID NOs: 19-21. Forexample, the Shiga toxin effector polypeptide comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO:20.

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a fourth cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage siteat the carboxy terminus of its A1 fragment region. In certain furtherembodiments, the Shiga toxin effector polypeptide is not cytotoxic andthe molecular moiety is cytotoxic.

In certain embodiments of Embodiment Sets #1 to #3, the binding regionand Shiga toxin effector polypeptide are linked together, eitherdirectly or indirectly.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide is fused to the binding region, either directly orindirectly, such as, e.g., via a linker known to the skilled worker. Thebinding region and Shiga toxin effector polypeptide may be fused by aproteinaceous linker comprising one or more amino acids. For example,the linker may comprise, consist essentially of, or consist of an aminoacid sequence selected from GSTSGSGKPGSGEGS (SEQ ID NO:93),AHHSEDPSSKAPKAP (SEQ ID NO:95), SPSTPPTPSPSTPPA (SEQ ID NO: 181),EFPKPSTPPGSSGGAP (SEQ ID NO:90), and GSTSGSGKPGSGEGSTKG (SEQ ID NO:96).The binding region and the Shiga toxin effector polypeptide may beindirectly fused together by the presence of an intervening single aminoacid residue, such as, e.g., an alanine residue.

In certain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises at least one peptide and/or polypeptide. In certain furtherembodiments, the binding region is or comprises an immunoglobulin orimmunoglobulin-type binding region. In certain further embodiments, thebinding region comprising a polypeptide selected from the groupconsisting of: an autonomous V_(H) domain, single-domain antibodyfragment (sdAb), nanobody®, heavy chain-antibody domain derived from acamelid (V_(H)H or V_(H) domain fragment), heavy-chain antibody domainderived from a cartilaginous fish antibody (V_(H)H or V_(H) domainfragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality. In certainembodiments of Embodiment Sets #1 to #3, the binding region comprises apolypeptide selected from the group consisting of: autonomous V_(H)domain, single-domain antibody fragment (sdAb), nanobody®, heavychain-antibody domain derived from a camelid (V_(H)H or V_(H) domainfragment), heavy-chain antibody domain derived from a cartilaginous fishantibody (V_(H)H or V_(H) domain fragment), immunoglobulin new antigenreceptor (IgNAR), V_(NAR) fragment, single-chain variable fragment(scFv), antibody variable fragment (Fv), complementary determiningregion 3 fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide(FR3-CDR3-FR4), Fd fragment, and antigen-binding fragment (Fab). Incertain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises a polypeptide selected from the group consisting of: anautonomous V_(H) domain, single-domain antibody fragment (sdAb),nanobody®,heavy chain-antibody domain derived from a camelid (V_(H)H orV_(H) domain fragment), heavy-chain antibody domain derived from acartilaginous fish antibody (V_(H)H or V_(H) domain fragment),immunoglobulin new antigen receptor (IgNAR), V_(NAR) fragment,single-chain variable fragment (scFv), antibody variable fragment (Fv),Fd fragment, and antigen-binding fragment (Fab). In certain embodimentsof Embodiment Sets #1 to #3, the binding region comprises a single-chainvariable fragment (scFv). The binding region may comprise a polypeptideselected from the group consisting of: an autonomous V_(H) domain,single-domain antibody fragment (sdAb), nanobody®,heavy chain-antibodydomain derived from a camelid (V_(H)H or V_(H) domain fragment),heavy-chain antibody domain derived from a cartilaginous fish antibody(V_(H)H or V_(H) domain fragment), immunoglobulin new antigen receptor(IgNAR), V_(NAR) fragment, single-chain variable fragment (scFv),antibody variable fragment (Fv), complementary determining region 3fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fdfragment, and antigen-binding fragment (Fab). For example, thecell-targeting molecule of the present invention comprises a bindingregion comprising one or more of: an antibody variable fragment, asingle-domain antibody fragment, a single-chain variable fragment, a Fdfragment, an antigen-binding fragment, an autonomous VH domain, a V_(H)Hfragment derived from a camelid antibody, a heavy-chain antibody domainderived from a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor. In a further example, the bindingregion comprises a single-chain variable fragment and/or a V_(H)Hfragment derived from a camelid antibody. In yet a further example, thebinding region comprises a single-chain variable fragment. In yet afurther example, the binding region comprises a V_(H)H fragment derivedfrom a camelid antibody.

In certain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises an immunoglobulin binding region comprising at least oneheavy-chain variable domain polypeptide linked to at least onelight-chain variable domain polypeptide by a linker comprising anon-branched sequence of thirteen or more amino acid residues,optionally wherein the linker comprises an amino acid sequence selectedfrom any one of (G₄S)₃ (SEQ ID NO: 180), (G₄S)₄ (SEQ ID NO: 177), (G₄S)₅(SEQ ID NO:92), (G₄S)₆ (SEQ ID NO: 178), or (G₄S)₇ (SEQ ID NO: 179).

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptideand/or capable of exhibiting a significant level of intracellularrouting activity to the endoplasmic reticulum and/or cytosol from anendosomal starting location of a cell.

For certain embodiments of Embodiment Sets #1 to #3, wherebyadministration of the cell-targeting molecule of the present inventionto a cell physically coupled with the extracellular target biomoleculeof the cell-targeting molecule’s binding region, the cell-targetingmolecule is capable of causing death of the cell. For certain furtherembodiments, administration of the cell-targeting molecule of theinvention to two different populations of cell types which differ withrespect to the presence or level of the extracellular targetbiomolecule, the cell-targeting molecule is capable of causing celldeath to the cell-types physically coupled with an extracellular targetbiomolecule of the cytotoxic cell-targeting molecule’s binding region ata CD₅₀ at least three times or less than the CD₅₀ to cell types whichare not physically coupled with an extracellular target biomolecule ofthe cell-targeting molecule’s binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first population of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule’s binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule’sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule’s binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule comprises a molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. In certainembodiments, the molecular moiety comprises or consists of the bindingregion. In certain embodiments, the molecular moiety comprises at leastone amino acid and the Shiga toxin effector polypeptide is linked to atleast one amino acid residue of the molecular moiety. In certain furtherembodiments, the molecular moiety and the Shiga toxin effectorpolypeptide are fused forming a continuous polypeptide.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule further comprises a cytotoxic molecular moiety associated withthe carboxy-terminus of the Shiga toxin effector polypeptide. Forcertain embodiments, the cytotoxic molecular moiety is a cytotoxicagent, such as, e.g., a small molecule chemotherapeutic agent,anti-neoplastic agent, cytotoxic antibiotic, alkylating agent,antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor knownto the skilled worker and/or described herein. For certain furtherembodiments, the cytotoxic molecular moiety is cytotoxic atconcentrations of less than 10,000, 5,000, 1,000, 500, or 200 pM.

In certain embodiments of Embodiment Sets #1 to #3, the binding regionis linked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is an immunoglobulin orimmunoglobulin-type binding region. For example, in the cell-targetingmolecule of the present invention, the binding region and the Shigatoxin effector polypeptide may be fused forming a continuous polypeptidesuch that the binding region is associated with the carboxy-terminus ofthe Shiga toxin A subunit effector polypeptide

In certain embodiments of Embodiment Sets #1 to #3, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned (1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ IDNO:1), Shiga toxin (SEQ ID NO:2), or another Shiga toxin 1 variantsequence (e.g. SEQ ID NOs: 4-6); or (2) at 247-250 of the A Subunit ofShiga-like toxin 2 (SEQ ID NO:3) or a Shiga-like toxin 2 variantsequence (e.g. SEQ ID NOs: 7-18); or the equivalent amino acid sequenceposition in any Shiga toxin A Subunit. In certain further embodiments,the mutation is an amino acid residue substitution of an arginineresidue with a non-positively charged, amino acid residue. In certainembodiments, the Shiga toxin effector polypeptide comprises a disruptedfurin-cleavage motif at the carboxy-terminus of the Shiga toxin A1fragment derived region, wherein said disrupted furin-cleavage motifcomprises (i) a carboxy-terminal truncation of as compared to thecarboxy-terminus of a wild-type Shiga toxin A Subunit and (ii) at leastone amino acid substitution in the furin-cleavage site relative to awild-type Shiga toxin A Subunit, at the natively positioned amino acidresidues 248 and 251 of the A Subunit of Shiga-like toxin 1 (SEQ IDNO:1), Shiga toxin (SEQ ID NO:2), or another Shiga toxin 1 effectorpolypeptide variant (SEQ ID NOs: 4-6); or at the natively positionedamino acid residues 247 and 250 of the A Subunit of Shiga-like toxin 2(SEQ ID NO:3) or a Shiga-like toxin 2 effector polypeptide variant (SEQID NOs: 7-18). In certain embodiments, the disrupted furin-cleavagemotif comprises a carboxy-terminal truncation as compared to a wild-typeShiga toxin A Subunit; and an amino acid substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit, atthe natively positioned amino acid residues 248 and 251 of the A Subunitof Shiga-like toxin 1 (SEQ ID NO:1) or Shiga toxin (SEQ ID NO:2); or atthe natively positioned amino acid residues 247 and 250 of the A Subunitof Shiga-like toxin 2 (SEQ ID NO:3). In certain embodiments, thesubstitution of the amino acid residue in the furin-cleavage motif is ofan arginine residue with an alanine residue.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention comprises the Shiga toxin effectorpolypeptide comprising or consisting essentially of the polypeptideshown in any one of SEQ ID NOs: 19-21 and 75-89.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a third cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment.

In certain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises the peptide or polypeptide shown in any one of SEQ ID NOs:45-74, 91-92, or 94.

In certain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceof: amino acids 269 to 501 of SEQ ID NO:24; amino acids 269 to 513 ofSEQ ID NO:25; amino acids 269 to 499 of SEQ ID NO:26; amino acids 269 to500 of SEQ ID NO:27; amino acids 269-520 of SEQ ID NO:28; amino acids269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; amino acids 268 to 386 ofSEQ ID NO:31; amino acids 269 to 499 of SEQ ID NO:32; amino acids 269 to499 of SEQ ID NO:33; amino acids 253 to 370 of SEQ ID NO:34; amino acids253 to 367 of SEQ ID NO:35; amino acids 269 to 514 of SEQ ID NO:36;amino acids 268 to 500 of SEQ ID NO:97; amino acids 268 to 512 of SEQ IDNO:98; amino acids 268 to 498 of SEQ ID NO:99; amino acids 268 to 499 ofSEQ ID NO: 100; amino acids 268-519 of SEQ ID NO: 101; or amino acids268 to 518 of SEQ ID NO: 102 or SEQ ID NO: 103. In certain embodiments,the binding region comprises, consists essentially of, or consists of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence of: amino acids 269 to 501 of SEQ ID NO:24; amino acids269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ ID NO:26;amino acids 269 to 500 of SEQ ID NO:27; amino acids 269-520 of SEQ IDNO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; aminoacids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ IDNO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; or amino acids 269to 514 of SEQ ID NO:36. In certain embodiments, the binding regioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceof: amino acids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 ofSEQ ID NO:26; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30;amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ IDNO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; or amino acids 269to 514 of SEQ ID NO:36.

In certain embodiments of Embodiment Sets #1 to #3, the binding regioncomprises the peptide or polypeptide shown in any one of SEQ ID NOs:45-74 and 90-96.

In certain embodiments of Embodiment Set #1 to #3, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any of the following: amino acids 269 to 501 of SEQ IDNO:24; amino acids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 ofSEQ ID NO:26; amino acids 269 to 500 of SEQ ID NO:27; amino acids269-520 of SEQ ID NO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQID NO:30; amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499of SEQ ID NO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253to 370 of SEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; aminoacids 269 to 514 of SEQ ID NO:36; amino acids 268 to 500 of SEQ IDNO:97; amino acids 268 to 512 of SEQ ID NO:98; amino acids 268 to 498 ofSEQ ID NO:99; amino acids 268 to 499 of SEQ ID NO: 100; amino acids268-519 of SEQ ID NO: 101; and amino acids 268 to 518 of SEQ ID NO: 102or SEQ ID NO: 103. In certain embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byany of the following: amino acids 269 to 501 of SEQ ID NO:24; aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ IDNO:26; amino acids 269 to 500 of SEQ ID NO:27; amino acids 269-520 ofSEQ ID NO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30;amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ IDNO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; and amino acids269 to 514 of SEQ ID NO:36. In certain embodiments, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any of the following: amino acids 269 to 513 of SEQ IDNO:25; amino acids 269 to 499 of SEQ ID NO:26; amino acids 269 to 519 ofSEQ ID NO:29 or SEQ ID NO:30; amino acids 268 to 386 of SEQ ID NO:31;amino acids 269 to 499 of SEQ ID NO 2; amino acids 269 to 499 of SEQ IDNO:33; amino acids 253 to 370 of SEQ ID NO:34; amino acids 253 to 367 ofSEQ ID NO:35; and amino acids 269 to 514 of SEQ ID NO:36. In certainembodiments, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 269 to 519 of SEQID NO:29, amino acids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370of SEQ ID NO:34; or amino acids 253 to 367 of SEQ ID NO:35. In certainembodiments, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 269 to 519 of SEQID NO:29. In certain, embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byamino acids 268 to 386 of SEQ ID NO:31. In certain, embodiments, thebinding region comprises, consists essentially of, or consists of thepolypeptide represented by amino acids 253 to 370 of SEQ ID NO:34. Incertain embodiments, the binding region comprises, consists essentiallyof, or consists of the polypeptide represented by amino acids 253 to 367of SEQ ID NO:35.

In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of the polypeptide shown in any one of SEQ ID NOs: 22-36 and97-108. In certain embodiments, the cell-targeting molecule of thepresent invention comprises, consists essentially of, or consists of thepolypeptide shown in any one of SEQ ID NOs: 25-27 and 29-36. In certainembodiments, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of the polypeptide shownin any one of SEQ ID NOs: 29, 31, 34 and 35. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO:29. Incertain embodiments, the cell-targeting molecule of the presentinvention comprises, consists essentially of, or consists of thepolypeptide shown in SEQ ID NO:31. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO:34. Incertain embodiments, the cell-targeting molecule of the presentinvention comprises, consists essentially of, or consists of thepolypeptide shown in SEQ ID NO:35. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO: 102.

In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence shown in any one of SEQ ID NOs:22-36 and 97-108. In certain embodiments of Embodiment Set #1 to #3, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more) identical to the amino acid sequence shown in any one of SEQ IDNOs: 25-27 and 29-36. In certain embodiments of Embodiment Set #1 to #3,the cell-targeting molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more) identical to the amino acid sequence shown in any one of SEQ IDNOs: 29, 31, 34 and 35. In certain embodiments of Embodiment Set #1 to#3, the cell-targeting molecule of the present invention comprises,consists essentially of, or consists of an amino acid sequence that isat least 85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more) identical to the amino acid sequence of SEQ ID NO: 29.In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence of SEQ ID NO:31. In certainembodiments of Embodiment Set #1 to #3, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofan amino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence of SEQ ID NO:34. In certain embodiments of Embodiment Set#1 to #3, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence ofSEQ ID NO:35.

In certain embodiments of Embodiment Set #1 to #3, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any of the following: amino acids 269 to 501 of SEQ IDNO:24; amino acids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 ofSEQ ID NO:26; amino acids 269 to 500 of SEQ ID NO:27; amino acids269-520 of SEQ ID NO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQID NO:30; amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499of SEQ ID NO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253to 370 of SEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; aminoacids 269 to 514 of SEQ ID NO:36; amino acids 268 to 500 of SEQ IDNO:97; amino acids 268 to 512 of SEQ ID NO:98; amino acids 268 to 498 ofSEQ ID NO:99; amino acids 268 to 499 of SEQ ID NO: 100; amino acids268-519 of SEQ ID NO: 101; amino acids 268 to 518 of SEQ ID NO: 102 orSEQ ID NO: 103; amino acids 267 to 384 of SEQ ID NO: 104; amino acids268 to 498 of SEQ ID NO: 105; amino acids 252 to 370 of SEQ ID NO: 106;amino acids 252 to 366 of SEQ ID NO: 107; and amino acids 268 to 513 ofSEQ ID NO: 108. In certain embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byany of the following: amino acids 269 to 501 of SEQ ID NO:24; aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ IDNO:26; amino acids 269 to 500 of SEQ ID NO:27; amino acids 269-520 ofSEQ ID NO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30;amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ IDNO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; and amino acids269 to 514 of SEQ ID NO:36. In certain embodiments, the binding regioncomprises, consists essentially of, or consists of the polypeptiderepresented by any of the following: amino acids 269 to 513 of SEQ IDNO:25; amino acids 269 to 499 of SEQ ID NO:26; amino acids 269 to 519 ofSEQ ID NO:29 or SEQ ID NO:30; amino acids 268 to 386 of SEQ ID NO:31;amino acids 269 to 499 of SEQ ID NO:32; amino acids 269 to 499 of SEQ IDNO:33; amino acids 253 to 370 of SEQ ID NO:34; amino acids 253 to 367 ofSEQ ID NO:35; and amino acids 269 to 514 of SEQ ID NO:36. In certainembodiments, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 269 to 519 of SEQID NO:29, amino acids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370of SEQ ID NO:34; or amino acids 253 to 367 of SEQ ID NO:35. In certainembodiments, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 269 to 519 of SEQID NO:29. In certain, embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byamino acids 268 to 386 of SEQ ID NO:31. In certain, embodiments, thebinding region comprises, consists essentially of, or consists of thepolypeptide represented by amino acids 253 to 370 of SEQ ID NO:34. Incertain embodiments, the binding region comprises, consists essentiallyof, or consists of the polypeptide represented by amino acids 253 to 367of SEQ ID NO:35.

In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of the polypeptide shown in any one of SEQ ID NOs: 22-36 and97-108. In certain embodiments, the cell-targeting molecule of thepresent invention comprises, consists essentially of, or consists of thepolypeptide shown in any one of SEQ ID NOs: 25-27 and 29-36. In certainembodiments, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of the polypeptide shownin any one of SEQ ID NOs: 29, 31, 34 and 35. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO:29. Incertain embodiments, the cell-targeting molecule of the presentinvention comprises, consists essentially of, or consists of thepolypeptide shown in SEQ ID NO:31. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of the polypeptide shown in SEQ ID NO:34. Incertain embodiments, the cell-targeting molecule of the presentinvention comprises, consists essentially of, or consists of thepolypeptide shown in SEQ ID NO:35.

In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence shown in any one of SEQ ID NOs:22-36 and 97-108. In certain embodiments of Embodiment Set #1 to #3, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more) identical to the amino acid sequence shown in any one of SEQ IDNOs: 25-27 and 29-36. In certain embodiments of Embodiment Set #1 to #3,the cell-targeting molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more) identical to the amino acid sequence shown in any one of SEQ IDNOs: 29, 31, 34 and 35. In certain embodiments of Embodiment Set #1 to#3, the cell-targeting molecule of the present invention comprises,consists essentially of, or consists of an amino acid sequence that isat least 85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more) identical to the amino acid sequence of SEQ ID NO: 29.In certain embodiments of Embodiment Set #1 to #3, the cell-targetingmolecule of the present invention comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence of SEQ ID NO:31. In certainembodiments of Embodiment Set #1 to #3, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofan amino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence of SEQ ID NO:34. In certain embodiments of Embodiment Set#1 to #3, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence ofSEQ ID NO:35.

In certain embodiments of Embodiment Sets #1 to #3, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Sets #1 to #3, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater, as long as the cell-targeting molecule retainsthe appropriate level of the Shiga toxin biological activity notedherein (e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Sets #1 to #3, the binding regionis comprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention comprises or consists essentially ofthe polypeptide shown in any one of SEQ ID NOs: 22-37 and 97-108, andoptionally the cell-targeting molecule comprises an amino-terminalmethionine residue.

For certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule of the present invention exhibits low cytotoxic potency (i.e.is not capable when introduced to certain positive target cell types ofexhibiting a cytotoxicity greater than 1% cell death of a cellpopulation at a cell-targeting molecule concentration of 1000 nM, 500nM, 100 nM, 75 nM, or 50 nM) and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a fifth cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the fifth cell-targeting molecule. In certain furtherembodiments, the fifth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Sets #1 to #3, the molecular moietycomprises a peptide and/or polypeptide derived from the Shiga toxin A2fragment of a naturally occurring Shiga toxin.

The embodiments of the present invention are not intended to cover anynaturally-occurring Shiga holotoxin or Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #1 to #3, the cell-targeting molecule ofthe present invention does not comprise a naturally occurring Shigatoxin B Subunit. In certain further embodiments, the cell-targetingmolecule of the invention does not comprise any polypeptide comprising,consisting essentially of, or consisting of a functional binding domainof a native Shiga toxin B subunit. Rather, in certain embodiments of thecell-targeting molecules of the invention, the Shiga toxin A Subunitderived regions are functionally associated with heterologous bindingregions to effectuate cell-targeting.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises at least two, embedded or inserted,heterologous epitopes.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide does not comprise the set of amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit selectedfrom the following sets: (1) R248H and R251H; (2) R248G and R251G; (3)A246G, S247A, A253G, and S254A; and (4) A246G, S247A, R248G, R251G,A253G, and S254A.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide does not comprise a deletion of the region nativelypositioned at 247-252 in a wild-type Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #1 to #3, the Shiga toxin effectorpolypeptide does not comprise deletions of the regions nativelypositioned at 245-247 and 253-255 in a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide comprises one or more mutations relative to anaturally occurring (or wild-type) A Subunit of a member of the Shigatoxin family which changes an enzymatic activity of the Shiga toxineffector polypeptide, the mutation selected from at least one amino acidresidue deletion, insertion, or substitution. In certain furtherembodiments, the mutation relative to the naturally occurring A Subunitreduces or eliminates a cytotoxic activity of the Shiga toxin effectorpolypeptide but the Shiga toxin effector polypeptide retains at leastone other Shiga toxin effector function, such as, e.g., promotingcellular internalization and/or directing intracellular routing to acertain subcellular compartment(s). In certain further embodiments, themutation relative to the naturally occurring (or wild-type) A Subunit isselected from at least one amino acid residue substitution, such as,e.g., A231E, R75A, Y77S, Y114S, E167D, R170A, R176K, and/or W203A in SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

For certain embodiments of Embodiment Sets #1 to #3, the Shiga toxineffector polypeptide is capable of: (i) routing to a subcellularcompartment of a cell in which the Shiga toxin effector polypeptide ispresent selected from the following: cytosol, endoplasmic reticulum, andlysosome; (ii) intracellular delivery of the epitope from an earlyendosomal compartment to a proteasome of a cell in which the Shiga toxineffector polypeptide is present; and/or (iii) intracellular delivery ofthe epitope to a MHC class I molecule from an early endosomalcompartment of a cell in which the Shiga toxin effector polypeptide ispresent. In certain further embodiments, the Shiga toxin effectorpolypeptide is capable of intracellular delivery of the CD 8+ T-cellepitope for presentation by a MHC class I molecule on the surface of acell in which the Shiga toxin effector polypeptide is present.

In certain embodiments, the molecule of the present invention does notcomprise, at a position carboxy-terminal of the Shiga toxin effectorpolypeptide and/or the carboxy-terminus of the Shiga toxin A1 fragmentregion, any additional exogenous material representing an antigen and/orheterologous, CD8+, T-cell epitope-peptide.

In certain embodiments of Embodiment Sets #1 to #3, the binding regiondoes not comprise a ligand.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule is de-immunized due to the embedded or inserted, heterologous,epitope, and exhibits reduced relative antigenicity and/or relativeimmunogenicity. The cell-targeting molecule exhibits reduced relativeantigenicity and/or relative immunogenicity as compared to a referencemolecule, such as, e.g., a seventh cell-targeting molecule consisting ofthe cell-targeting molecule except for it lacks one or more embedded orinserted epitopes present in the cell targeting molecule.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption, andexhibits reduced relative antigenicity and/or relative immunogenicity.The cell-targeting molecule exhibits reduced relative antigenicityand/or relative immunogenicity as compared to a reference cell-targetingmolecule consisting of the cell-targeting molecule except for thefurin-cleavage motif is wild-type and/or all the Shiga toxin effectorpolypeptide components consist of a wild-type Shiga toxin A1 fragment.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule is de-immunized due to the plurality of disrupted B-cell and/orCD4+ T-cell epitope regions and exhibits reduced relative B-cell and/orCD4+ T-cell antigenicity and/or reduced relative B-cell and/or CD4+T-cell immunogenicity. In certain further embodiments, thecell-targeting molecule exhibits reduced relative B-cell antigenicityand/or relative B-cell immunogenicity as compared to a referencemolecule, such as, e.g., a wild-type Shiga toxin A1 fragment orcell-targeting molecule comprising the aforementioned, such as a thirdcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment. In certain furtherembodiments, the cell-targeting molecule exhibits reduced relative CD4+T-cell antigenicity and/or relative CD4+ T-cell immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the Shiga toxin effector polypeptidecomponent(s) comprises a wild-type Shiga toxin A1 fragment sequence.

In certain embodiments of Embodiment Sets #1 to #3, the cell-targetingmolecule is in the form of a pharmaceutically acceptable salt orsolvate. Among certain embodiments of the present invention is apharmaceutical composition comprising any one of the above Shiga toxineffector polypeptides of the present invention and/or any one of theabove cell-targeting molecules of the present invention; and at leastone pharmaceutically acceptable excipient or carrier. The at least onepharmaceutically acceptable carrier may include a solvent, a dispersionmedium, a coating, an antimicrobial agent, an isotonic agent, or anabsorption delaying agent; and/or wherein the pharmaceutical compositionfurther comprises an aqueous or non-aqueous carrier; a surfactant; astabilizer, a preservative, a buffer, an antioxidant, a wetting agent,an emulsifying agent, a dispersing agent; an isotonic agent; and/or anantibacterial or antifungal agent.

Among certain embodiments of the present invention is a diagnosticcomposition comprising any one of the above cell-targeting molecules ofthe present invention and a detection promoting agent. Certain furtherembodiments are cell-targeting molecules of the present inventionwherein the detection promoting agent is a heterologous epitope and thecell-targeting molecule comprises the heterologous epitope.

Beyond the Shiga toxin effector polypeptides of the present invention,cell-targeting molecules of the present invention, and compositionsthereof, polynucleotides capable of encoding a cell-targeting moleculeof the present invention are within the scope of the present invention,as well as expression vectors which comprise a polynucleotide of thepresent invention and host cells comprising any polynucleotide and/orexpression vector of the present invention. Host cells comprising anexpression vector may be used, e.g., in methods for producing a moleculeof the present invention or a polypeptide component or fragment thereofby recombinant expression.

Among certain embodiments of the present invention is a method ofkilling a cell (e.g. a HER2-expressing cell), the method comprising thestep of contacting the cell with any of the above cell-targetingmolecules of the present invention or the above pharmaceuticalcompositions of the present invention. In certain embodiments, the stepof contacting the cell(s) occurs in vitro. In certain embodiments, thecell expresses muc-4 and/or CD44. In certain embodiments, the cell isresistant to cytotoxicity caused by T-DM1 (trastuzumab emtansine) and/ortrastuzumab. In further embodiments of the cell-killing methods, themethod is capable of selectively killing cell(s) and/or cell typespreferentially over other cell(s) and/or cell types when contacting amixture of cells which differ with respect to the extracellular presenceand/or expression level of a target HER2/neu/ErbB2 of the binding regionof the cell-targeting molecule. In certain further embodiments thecell(s) are in the presence of pertuzumab, T-DM1 (trastuzumabemtansine), and/or lapatinib and/or had previously been contacted withpertuzumab, T-DM1 (trastuzumab emtansine), and/or lapatinib. In certainembodiments, the step of contacting the cell(s) occurs or in vivo. Infurther embodiments of the cell-killing methods, the method is capableof selectively killing cell(s) and/or cell types preferentially overother cell(s) and/or cell types when contacting a mixture of cells whichdiffer with respect to the extracellular presence and/or expressionlevel of an extracellular target biomolecule of the binding region ofthe cell-targeting molecule.

Among certain embodiments of the present invention is a method ofkilling a cell (e.g. a HER2-expressing cell), the method comprising thestep of contacting the cell with any of the above cell-targetingmolecules of the present invention or the above pharmaceuticalcompositions of the present invention. In certain embodiments, the stepof contacting the cell(s) occurs in vitro. In certain other embodiments,the step of contacting the cell(s) occurs or in vivo. In certainembodiments, the cell expresses muc-4 and/or CD44. In certainembodiments, the cell is resistant to cytotoxicity caused by T-DM1(trastuzumab emtansine) and/or trastuzumab. In further embodiments ofthe cell-killing methods, the method is capable of selectively killingcell(s) and/or cell types preferentially over other cell(s) and/or celltypes when contacting a mixture of cells which differ with respect tothe extracellular presence and/or expression level of an extracellulartarget biomolecule of the binding region of the cell-targeting molecule.In certain further embodiments the cell(s) are in the presence ofpertuzumab, T-DM1 (trastuzumab emtansine), and/or lapatinib and/or hadpreviously been contacted with pertuzumab, T-DM1 (trastuzumabemtansine), and/or lapatinib.

Among certain embodiments of the present invention is a method ofkilling a cell (e.g. a HER2-expressing cell), the method comprising thestep of contacting the cell with any of the above cell-targetingmolecules of the present invention or the above pharmaceuticalcompositions of the present invention wherein the cell is in thepresence of pertuzumab, T-DM1 (trastuzumab emtansine), and/or lapatiniband/or had previously been contacted with pertuzumab, T-DM1 (trastuzumabemtansine), and/or lapatinib. In certain embodiments, the step ofcontacting the cell(s) occurs in vitro. In certain other embodiments,the step of contacting the cell(s) occurs or in vivo. In certainembodiments, the cell expresses muc-4 and/or CD44. In certainembodiments, the cell is resistant to cytotoxicity caused by T-DM1(trastuzumab emtansine) and/or trastuzumab. In further embodiments ofthe cell-killing methods, the method is capable of selectively killingcell(s) and/or cell types preferentially over other cell(s) and/or celltypes when contacting a mixture of cells which differ with respect tothe extracellular presence and/or expression level of an extracellulartarget biomolecule of the binding region of the cell-targeting molecule.

The present invention further provides methods of treating diseases,disorders, and/or conditions in patients, the methods each comprisingthe step of administering to a patient in need thereof a therapeuticallyeffective amount of a cell-targeting molecule of the present inventionand/or pharmaceutical composition of the present invention. For certainembodiments, the method of treating diseases, disorders, and/orconditions in a patient in need thereof further comprises administeringto the patient in need thereof a therapeutically effective amount of oneor more additional HER2-targeting therapeutic agent as described herein.For certain embodiments, the patient in need thereof has been previouslytreated with one or more additional HER2-targeting therapeutic agentand/or does not respond to, or does not benefit from, treatment with oneor more additional HER2-targeting therapeutic agent. For certainembodiments, the disease, disorder, or condition to be treated using amethod of the invention is selected from: a cancer, tumor, growthabnormality, immune disorder, or microbial infection. In certainembodiments of these methods, the cancer to be treated is selected fromthe group consisting of: bone cancer, breast cancer, central/peripheralnervous system cancer, gastrointestinal cancer, germ cell cancer,glandular cancer, head-neck cancer, hematological cancer, kidney-urinarytract cancer, liver cancer, lung/pleura cancer, prostate cancer,sarcoma, skin cancer, and uterine cancer, such as, e.g., breast cancer,gastric cancer, urothelial cancer, bladder cancer, urothelial bladdercancer, serous uterine cancer, extrahepatic biliary tract cancer, orbiliary carcinoma. For certain embodiments, the cancer being treated isbreast cancer and/or gastrointestinal cancer. For certain embodiments ofthese methods, the immune disorder to be treated is an immune disorderassociated with a disease selected from the group consisting of:amyloidosis, ankylosing spondylitis, asthma, Crohn’s disease, diabetes,graft rejection, graft-versus-host disease, Hashimoto’s thyroiditis,hemolytic uremic syndrome, HIV-related disease, lupus erythematosus,multiple sclerosis, polyarteritis nodosa, polyarthritis, psoriasis,psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock,Sjögren’s syndrome, ulcerative colitis, and vasculitis.

The use of any composition of matter of the present invention for thetreatment or prevention of a cancer, tumor, growth abnormality, and/orimmune disorder is within the scope of the present invention. Amongcertain embodiments of the present invention is a cell-targetingmolecule of the present invention and/or a pharmaceutical composition ofthe invention for use in the treatment or prevention of a disease,disorder or condition in a patient in need thereof. Furthermore, thediagnostic composition, polynucleotide, expression vector, and host cellof the present invention are for use in the treatment or prevention of adisease, disorder or condition in a patient in need thereof. Amongcertain embodiments of the present invention is a cell-targetingmolecule of the present invention and/or a pharmaceutical compositionthereof for the treatment or prevention of a cancer, tumor, growthabnormality, immune disorder, and/or microbial infection. Among certainembodiments of the present invention is the use of a cell-targetingmolecule of the present invention and/or pharmaceutical composition ofthe present invention in the manufacture of a medicament for thetreatment or prevention of a disease, disorder or condition in a patientin need thereof. Furthermore, the present invention provides the use ofthe diagnostic composition, polynucleotide, expression vector, and hostcell of the present invention in the manufacture of a medicament for thetreatment or prevention of a disease, disorder or condition in a patientin need thereof. Among certain embodiments of the present invention isthe use of a cell-targeting molecule of the present invention and/orpharmaceutical composition thereof in the manufacture of a medicamentfor the treatment or prevention of a cancer, tumor, growth abnormality,immune disorder, or microbial infection. Furthermore, the presentinvention provides the use of the diagnostic composition,polynucleotide, expression vector, and host cell of the presentinvention in the manufacture of a medicament for the treatment orprevention of a cancer, tumor, growth abnormality, immune disorder, ormicrobial infection. The “disease, disorder or condition” or the“cancer, tumor, growth abnormality, immune disorder, or microbialinfection” may be characterized by cells that are physically coupledwith HER2/neu/ErbB2. The HER2/neu/ErbB2 target biomolecule can bephysically coupled to the surface of the cells. In certain embodiments,the disease, disorder or condition may be characterized by cells thatexpress the HER2/neu/ErbB2 target biomolecule (including cells thatoverexpress HER2). The HER2/neu/ErbB2 can be expressed (includingoverexpressed) at the surface of the cells.

Certain embodiments of the cell-targeting molecules of the presentinvention may be utilized for the delivery of additional exogenousmaterial into a cell physically coupled with an extracellular targetbiomolecule of the cell-targeting molecule of the present invention.Additionally, the present invention provides a method for deliveringexogenous material to the inside of a cell(s) comprising contacting thecell(s), either in vitro or in vivo, with a cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention. The present invention further provides a method fordelivering exogenous material to the inside of a cell(s) (e.g. aHER2-expressing cell) in a patient, the method comprising the step ofadministering to the patient a cell-targeting molecule of the presentinvention (with or without cytotoxic activity), wherein the targetcell(s) is physically coupled with an extracellular target biomoleculeof the cell-targeting molecule.

Among certain embodiments of the present invention is a method ofdelivering into a cell (e.g. a HER2-expressing cell), the method aT-cell epitope capable of being presented by a MHC class I molecule ofthe cell, the method comprising the step of contacting the cell with thecell-targeting molecule of the present invention which is associatedwith a heterologous, T-cell epitope and/or a composition thereof (e.g.,a pharmaceutical or diagnostic composition of the present invention).

Among certain embodiments of the present invention is a method for“seeding” a tissue locus within a chordate, the method comprising thestep of: administering to the chordate a cell-targeting molecule of thepresent invention, a pharmaceutical composition of the presentinvention, and/or a diagnostic composition of the present invention (seee.g. WO 2017/019623; WO 2018/140427). For certain further embodiments,the methods of the invention for “seeding” a tissue locus are for“seeding” a tissue locus which comprises a malignant, diseased, orinflamed tissue. The malignant, diseased, or inflamed tissue may becharacterized by cells that are physically coupled with HER2/neu/ErbB2.The HER2/neu/ErbB2 target biomolecule can be physically coupled to thesurface of the cells. For certain embodiments, the disease, disorder orcondition may be characterized by cells that express the HER2/neu/ErbB2target biomolecule (including cells that overexpress HER2). TheHER2/neu/ErbB2 can be expressed (including overexpressed) at the surfaceof the cells. For certain further embodiments, the methods of theinvention for “seeding” a tissue locus are for “seeding” a tissue locuswhich comprises the tissue selected from the group consisting of:diseased tissue, tumor mass, cancerous growth, tumor, infected tissue,or abnormal cellular mass. For certain further embodiments, the methodsof the invention for “seeding” a tissue locus comprises administering tothe chordate the cell-targeting molecule of the invention, thepharmaceutical composition of the invention, or the diagnosticcomposition of the invention comprising the heterologous, T-cell epitopeselected from the group consisting of: peptides not natively presentedby the target cells of the cell-targeting molecule in MHC class Icomplexes, peptides not natively present within any protein expressed bythe target cell, peptides not natively present within the proteome ofthe target cell, peptides not natively present in the extracellularmicroenvironment of the site to be seeded, and peptides not nativelypresent in the tumor mass or infected tissue site to be targeted. Thediseased tissue, tumor mass, cancerous growth, tumor, infected tissue,or abnormal cellular mass may be characterized by cells that arephysically coupled with HER2/neu/ErbB2. The HER2/neu/ErbB2 targetbiomolecule can be physically coupled to the surface of the cells. Forcertain embodiments, the disease, disorder or condition may becharacterized by cells that express the HER2/neu/ErbB2 targetbiomolecule (including cells that overexpress HER2). The HER2/neu/ErbB2can be expressed (including overexpressed) at the surface of the cells.

The use of any composition of matter of the present invention for thediagnosis, prognosis, and/or characterization of a disease, disorder,and/or condition is within the scope of the present invention. Forexample, the use of the cell-targeting molecule, pharmaceuticalcomposition, diagnostic composition, polynucleotide, expression vector,and host cell of the present invention for the diagnosis, prognosis,and/or characterization of a disease, disorder, and/or condition iswithin the scope of the present invention. Among certain embodiments ofthe present invention is a method of using a cell-targeting molecule ofthe present invention comprising a detection promoting agent and/orcomposition of the present invention (e.g. a diagnostic composition) forthe collection of information useful in the diagnosis, prognosis, orcharacterization of a disease, disorder, or condition. Among certainembodiments of the present invention is the method of detecting a cell(or subcellular compartment thereof) using a cell-targeting moleculeand/or diagnostic composition of the present invention, the methodcomprising the steps of contacting a cell with the cell-targetingmolecule and/or diagnostic composition and detecting the presence ofsaid cell-targeting molecule and/or diagnostic composition. In certainembodiments, the step of contacting the cell(s) occurs in vitro. Incertain embodiments, the step of contacting the cell(s) occurs in vivo.In certain embodiments, the step of detecting the cell(s) occurs invitro. In certain embodiments, the step of detecting the cell(s) occursin vivo. In certain further embodiments, the method involves thedetection of the location of the cell-targeting molecule in an organismusing one or more imaging procedures after the administration of thecell-targeting molecule to said organism. For example, cell-targetingmolecules of the invention which incorporate detection promoting agentsas described herein may be used to characterize diseases as potentiallytreatable by a related pharmaceutical composition of the presentinvention. For example, certain cell-targeting molecules of the presentinvention and compositions thereof (e.g. pharmaceutical compositions anddiagnostic compositions of the present invention), and methods of thepresent invention may be used to determine if a patient belongs to agroup that responds to a pharmaceutical composition of the presentinvention. For example, certain cell-targeting molecules of the presentinvention and compositions thereof may be used to identify cells whichpresent a delivered heterologous epitope-peptide on a cellular surfaceand/or to identify subjects containing cells which present aheterologous epitope-peptide delivered by a cell-targeting molecule ofthe present invention. The “disease, disorder or condition” may becharacterized by cells that are physically coupled with HER2/neu/ErbB2.The HER2/neu/ErbB2 target biomolecule can be physically coupled to thesurface of the cells. In certain embodiments, the disease, disorder orcondition may be characterized by cells that express the HER2/neu/ErbB2target biomolecule (including cells that overexpress HER2). TheHER2/neu/ErbB2 can be expressed (including overexpressed) at the surfaceof the cells.

Among certain embodiments of the present invention is a method ofproducing a molecule of the present invention, the method comprising thestep of purifying the molecule of the present invention using abacterial cell-wall protein domain interaction, such as, e.g., protein Lfrom P. magnus or derivatives and binding domain fragments thereof orprotein A from S. aureus or derivatives and binding domain fragmentsthereof. For certain further embodiments, the purifying step of themethod involves the cell-targeting molecule comprising, consistingessentially of, or consisting of any one of the polypeptides shown inSEQ ID NOs: 22-36 or 97-108.

Among certain embodiments of the present invention are kits comprising acomposition of matter of the present invention, and optionally,instructions for use, additional reagent(s), and/or pharmaceuticaldelivery device(s). For example, the present invention provides a kitcomprising: (i) a cell-targeting molecule of the present invention, (ii)a pharmaceutical composition of the present invention, (iii) adiagnostic composition of the present invention, (iv) a polynucleotideof the present invention, (v) an expression vector of the presentinvention and/or (vi) a host cell of the present invention; andoptionally, instructions for use, additional reagent(s), and/orpharmaceutical delivery device(s). The kit may further comprise reagentsand other tools for detecting a cell type (e.g. a tumor cell) in asample or in a subject.

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures. Theaforementioned elements of the invention may be individually combined orremoved freely in order to make other embodiments of the invention,without any statement to object to such synthesis or removalhereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts exemplary HER2-targeting molecules comprising one or morede-immunized Shiga toxin A Subunit effector polypeptides and one or moreHER2 binding regions. These exemplary cell-targeting molecules eachcomprise a Shiga toxin effector polypeptide having de-immunizingmutations and a disrupted furin cleavage site near the carboxy terminusof the Shiga toxin effector polypeptide. A dashed, vertical, gray linedepicts a disrupted furin-cleavage site at the carboxy-terminus of an A1fragment derived region of the Shiga toxin effector polypeptide. The “N”and “C” denote an amino-terminus and carboxy-terminus, respectively, ofa polypeptide component of a cell-targeting molecule. In one exemplaryHER2-targeting molecule, the HER2 binding region is a scFv, and the scFvis shown participating in intermolecular variable domain exchange with aneighboring scFv (bottom left). The depictions of exemplary molecules inFIG. 1 are for illustrative purposes of certain, general arrangements ofthe structural features of a limited set of embodiments of the presentinvention. It is to be understood that these exemplary molecules do notintend, nor should any be construed, to be wholly definitive as to thearrangement of any structural features and/or components of a moleculeof the present invention. The relative size, location, or number offeatures shown in the schematics of FIG. 1 have been simplified. Theschematics in FIG. 1 are not intended to accurately portray anyinformation regarding the relative sizes of molecular structures in anyembodiment of the present invention.

FIG. 2 shows a Coomassie™-stained, sodium dodecyl sulfate,polyacrylamide gel (SDS-PAGE) after electrophoresis of samples ofexemplary HER2-targeting molecules 114778 (SEQ ID NO:24), 114795 (SEQ IDNO:25), 114791 (SEQ ID NO:26), and a molecular weight size marker, allprepared for gel-loading in reducing conditions. The samples of 114778(SEQ ID NO:24), 114795 (SEQ ID NO:25), and 114791 (SEQ ID NO:26) wereprepared using chromatography involving a chitin binding interaction andthen cleavage away from a chitin resin chromatography column by removalof the affinity tag (SEQ ID NO:43) and elution. FIG. 2 shows that thesizes of the predominant protein in the reduced samples of thepreparations of the molecules 114778 (SEQ ID NO:24), 114795 (SEQ IDNO:25), and 114791 (SEQ ID NO:26) were all about 55 kiloDaltons (kDa).

FIG. 3 graphically shows the results of a cell-kill assay investigatingthe activities of the HER2-targeting molecules 114778 (SEQ ID NO:24),114795 (SEQ ID NO:25), and 114791 (SEQ ID NO:26). FIG. 3 shows that theexemplary HER2-targeting molecules 114778 (SEQ ID NO:24), 114795 (SEQ IDNO:25), and 114791 (SEQ ID NO:26) exhibited cytotoxicity to two,different HER2-expressing cell-types: HCC1954 and NCI/ADR-RES-HER2+cells. The percent viability of target positive cells for each cell typewas plotted over the logarithm to base 10 of the HER2-targeting moleculeconcentrations administered to the respective cells.

FIG. 4 shows a Coomassie™-stained, SDS-PAGE gel after electrophoresis ofsamples of exemplary HER2-targeting molecules 114773 (SEQ ID NO:22) and114791 (SEQ ID NO:26), and a molecular weight size marker, all preparedfor gel-loading in non-reducing conditions. The samples of 114773 (SEQID NO:22) and 114791 (SEQ ID NO:26), both comprising a carboxy-terminalintein chitin binding domain (CBD) sequence (SEQ ID NO:43), wereprepared using Protein-L affinity chromatography. FIG. 4 shows that thesize of the predominant protein in the sample of the preparations of themolecules 114773 (SEQ ID NO:22) was about 100 kiloDaltons (kDa) whereasthe 114791 (SEQ ID NO:26) sample was devoid of protein signal in thisassay, presumably due to a lack in Protein L binding affinity.

FIG. 5 shows two Coomassie™-stained, SDS-PAGE gels after electrophoresisof samples of exemplary HER2-targeting molecules 114912 (SEQ ID NO:28),115111 (SEQ ID NO:29), 115411 (SEQ ID NO:30), and a molecular weightsize marker, all prepared for gel-loading in non-reducing conditions.The samples of 114912 (SEQ ID NO:28), 115111 (SEQ ID NO:29), 115411 (SEQID NO:30) were prepared using Protein-L affinity chromatography and notusing any chitin binding affinity tag. FIG. 5 shows that the size of thepredominant protein in the sample of the preparations of the molecules115111 (SEQ ID NO:29), 115411 (SEQ ID NO:30), and 114912 (SEQ ID NO:28)for each was about 55 kiloDaltons (kDa).

FIG. 6 graphically shows that the exemplary HER2-targeting molecules114912 (SEQ ID NO:28) and 115111 (SEQ ID NO:29) exhibited cytotoxicityto five different HER2-expressing cell-types: HCC1954,NCI/ADR-RES-HER2+, JIMT-1, SK-OV-3, and HCC1419 cells. The percentviability of cells was plotted over the logarithm to base 10 of theadministered HER2-targeting protein concentrations. FIG. 6 graphicallyshows that 115111 (SEQ ID NO:29) often had more potent cytotoxicity than114912 (SEQ ID NO:28). FIG. 6 also shows that for most of theHER2-targeting molecule concentrations tested no cytotoxicity wasobserved for MCF7 cells, which express very low levels of HER2. Thesamples of 114912 (SEQ ID NO:28) and 115111 (SEQ ID NO:29) were preparedusing Protein-L affinity chromatography without using any chitin bindingaffinity tag.

FIG. 7 graphically shows that the exemplary HER2-targeting molecules115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), 115194 (SEQ ID NO:33), and115195 (SEQ ID NO:32) exhibited cytotoxicity to four differentHER2-expressing cell-types: HCC1954, NCI/ADR-RES-HER2+, JIMT-1, andHCC1569 cells. The percent viability of cells was plotted over thelogarithm to base 10 of the administered HER2-targeting proteinconcentrations. FIG. 7 graphically shows that 115111 (SEQ ID NO:29),115172 (SEQ ID NO:23), 115194 (SEQ ID NO:33), and 115195 (SEQ ID NO:32)exhibited similar cytotoxic activities in this assay under theconditions tested. FIG. 6 also shows that no cytotoxicity was observedfor HER2 negative ST486 cells for most of the HER-targeting moleculeconcentrations tested.

FIG. 8 graphically shows the protein synthesis inhibition activities ofexemplary HER2-targeting molecules of the present invention in vitro andover a range of concentrations. For each sample molecule, theluminescent intensity of luciferase expressed during the assay inrelative luminescent units (RLU times e³) was plotted over the logarithmto base 10 of the concentration of the HER2-targeting molecule tested inpicomolar (pM). These exemplary HER2-targeting molecules 115111 (SEQ IDNO:29), 115172 (SEQ ID NO:23), and 115411 (SEQ ID NO:30) exhibitedribosome inhibition activities comparable to a “control” molecule, aShiga toxin effector polypeptide (SLTA-DI-2 (SEQ ID NO:20)) alone, notcoupled with any targeting agent or binding region. Additionally, theprotein synthesis inhibition activity of 115111 (SEQ ID NO:29) wassimilar to the activity of 115172 (SEQ ID NO:23), indicating that thesame scFv fused with either SLTA-DI-2 (SEQ ID NO:20) or SLTA-FR (SEQ IDNO:37), resulted in similar ribosomal inhibition activities.

FIG. 9 graphically shows that the exemplary HER2-targeting molecules115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), and 115411 (SEQ ID NO:30)exhibited cytotoxicity to NCI/ADR-RES-HER2+ cells. The percent viabilityof cells was plotted over the logarithm to base 10 of the administeredprotein concentrations. FIG. 9 also shows that an untargeted Shiga toxineffector polypeptide (SLTA-DI-2 (SEQ ID NO:20)) alone was not cytotoxicin this assay over the range of concentrations tested. FIG. 9graphically shows that 115111 (SEQ ID NO:29) and 115172 (SEQ ID NO:23)exhibited more potent cytotoxicity than 115411 (SEQ ID NO:30) at severalconcentrations.

FIG. 10 graphically shows HER2 binding characteristics of exemplaryHER2-targeting molecules of the present invention using HER2 positiveHCC1954 cells and a flow cytometry method. For each sample molecule, thefluorescence signal of FITC measured as mean fluorescent intensity(total MFI) was plotted over the logarithm to base 10 of theconcentration of the HER2-targeting molecule tested in µg/mL. Theexemplary HER2-targeting molecules 114912 (SEQ ID NO:28), 115111 (SEQ IDNO:29), 115195 (SEQ ID NO:32), 115645 (SEQ ID NO:34), and 115845 (SEQ IDNO:35) all exhibited binding to HER2 positive cells albeit with varyingcharacteristics. 115111 (SEQ ID NO:29), 115195 (SEQ ID NO:32), and115845 (SEQ ID NO:35) appeared to exhibit the highest affinity bindingto HCC1954 cells under the conditions in this assay.

FIG. 11 shows two pictorial representations of the human HER2 proteinstructure with certain residues marked for their involvement in beingbound by HER2 binding proteins. On the left side of FIG. 11 , the HER2residues involved in 115111 (SEQ ID NO:29) binding human HER2 are shownas red and blue atomic space filling spheres. On the right side of FIG.11 , the same HER2 residues are shown just as blue atomic space fillingspheres, the HER2 residues known to be critical for binding by certainapproved anti-HER2 therapeutic monoclonal antibodies: the HER2 residuesknown to be critical for binding by pertuzumab binding are shown asmagenta space filling spheres, and HER2 residues known to be criticalfor trastuzumab binding are shown as purple atomic space fillingspheres. FIG. 11 demonstrates that the HER2 epitope bound by 115111 (SEQID NO:29) was mapped within the HER2 extracellular domain (ECD) todomain I (on right in green); the HER2 epitope bound by pertuzumab wasmapped to domain II of the ECD, and the HER2 epitope bound bytrastuzumab was mapped to domain IV of the ECD. FIG. 11 highlights theHER2 epitopes bound by 115111 (SEQ ID NO:29), pertuzumab, andtrastuzumab are distinct and distant from each other.

FIG. 12 graphically shows the results of a human membrane proteome arrayassay used to test the specificity and selectivity of HER2 binding bythe exemplary HER2-targeting molecule 115111 (SEQ ID NO:29). The resultsshown in FIG. 12 show that only HER2 was identified and validated asbeing bound by 115111 (SEQ ID NO:29) from among about 5,300 differentproteins. Flow cytometry was used to identify the binding signal foreach individual protein, and data was normalized to background signal.Non-specific fluorescence was determined to be any value below threestandard deviations above noise (dotted line).

FIG. 13 graphically shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) is more cytotoxic than T-DM1 to two differentHER2-expressing cell-types: HCC1954 and NCI/ADR-RES-HER2+. The percentviability of cells was plotted over the logarithm to base 10 of theadministered HER2-targeting molecule concentrations. FIG. 13 also showsthat no cytotoxicity was observed for HER2 negative MDA-MB-468 cellscontacted with HER2-targeting molecule 115111 (SEQ ID NO:29) testedunder the conditions of the assay.

FIG. 14 graphically shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) is cytotoxic to two different HER2-expressingcell-types, HCC1419 and HCC1954, in either the presence or absence oflapatinib. The percent viability of cells was plotted for differentconditions, including: HCC1419 cells treated with lapatinib only at 1µM, 115111 (SEQ ID NO:29) at 20 nanogram per milliliter (ng/mL), andboth 115111 (SEQ ID NO:29) at 20 ng/mL and lapatinib at 1 µM; or HCC1954cells treated with lapatinib only at 1 µM, 115111 (SEQ ID NO:29) at 2ng/mL, and both 115111 (SEQ ID NO:29) at 2 ng/mL and lapatinib at 1 µM.FIG. 14 also shows a control treatment using the Shiga toxin effectorpolypeptide SLTA-DI-2 (SEQ ID NO:20) alone, which lacks any specifictargeting agent or binding region for cell-targeting, resulted in noalteration to cell viability in this assay.

FIG. 15 graphically shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) is cytotoxic to HER2 positive HCC1954 cells in thepresence of T-DM1. The cells were treated with 115111 (SEQ ID NO:29)alone T-DM1 alone, or both 115111 (SEQ ID NO:29) and T-DM1 mixedtogether at equal concentrations. The percent viability of cells wasplotted over the logarithm to base 10 of the total administered proteinconcentration: either 115111 (SEQ ID NO:29) alone, T-DM1 alone, or thetotal of both T-DM1 and 115111 (SEQ ID NO:29).

FIG. 16 graphically shows the activity of HER2-targeting molecules inthe presence of excess trastuzumab (20 µg/mL) pretreated for 1 hourprior to the addition of HER2-targeting molecules. The percent viabilityof HER2 positive HCC1954 cells was plotted over the logarithm to base 10of the administered HER2-targeting molecule concentrations. FIG. 16shows the exemplary HER2-targeting molecule 115111 (SEQ ID NO:29) iscytotoxic to cells in the presence of excess trastuzumab. The top graphof FIG. 16 shows that the exemplary HER2-targeting molecule 114912 (SEQID NO:28) was not cytotoxic in the presence of excess trastuzumab underthe conditions tested. The middle graph of FIG. 16 shows that theexemplary HER2-targeting molecule 115111 (SEQ ID NO:29) was cytotoxic toHCC1954 cells pre-incubated with excess trastuzumab, with no significantloss in cytotoxicity. The bottom graph shows that the cytotoxic activityof T-DM1 to HCC1954 cells was reduced by preincubation of the cells withexcess trastuzumab (20 µg/mL).

FIG. 17 graphically shows the cytotoxic activities of the exemplaryHER2-targeting molecule 115111 (SEQ ID NO:29) in the presence of excesstrastuzumab (100 µg/mL), pertuzumab (100 µg/mL), or both (100 µg/mL ofeach antibody), pretreated for 1 hour prior to the addition ofHER2-targeting molecules. The percent viability of HER2 positive cellswas plotted over the logarithm to base 10 of the administered 115111(SEQ ID NO:29) concentrations. FIG. 17 shows the exemplaryHER2-targeting molecule 115111 (SEQ ID NO:29) is cytotoxic to cells inthe presence of excess trastuzumab, pertuzumab, or both trastuzumab andpertuzumab. The top graph of FIG. 17 shows that the exemplaryHER2-targeting molecule 115111 (SEQ ID NO:29) was cytotoxic to HCC1954cells pre-incubated with excess trastuzumab, pertuzumab, or both. Thebottom graph shows that the exemplary HER2-targeting molecule 115111(SEQ ID NO:29) was cytotoxic to NCI-N87 cells pre-incubated with excesstrastuzumab, pertuzumab, or both. FIG. 17 also shows the cytotoxicity oftreatment of the cells with 115111 (SEQ ID NO:29) alone. Thecytotoxicity of 115111 (SEQ ID NO:29) alone appeared to be very similarto its cytotoxicity in the presence of excess trastuzumab, excesspertuzumab, or excess of both trastuzumab and pertuzumab.

FIG. 18 graphically shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) is more potently cytotoxic than exemplaryHER2-targeting molecule 114912 (SEQ ID NO:28) to HER2 expressing cellsfor shorter exposure durations. The percent viability of HER2 positiveSKBR3 cells was plotted over the logarithm to base 10 of theadministered HER2-targeting molecule concentrations. The top graph ofFIG. 18 shows that the exemplary HER2-targeting molecule 115111 (SEQ IDNO:29) was more cytotoxic to SKBR3 cells than 114912 (SEQ ID NO:28) athigher concentrations under the conditions of 1-hour exposures. Themiddle graph of FIG. 18 shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) was more cytotoxic to SKBR3 cells than 114912 (SEQID NO:28) at higher concentrations under the conditions of 4-hourexposures. The bottom graph shows that the exemplary HER2-targetingmolecules 115111 (SEQ ID NO:29) and 114912 (SEQ ID NO:28) exhibitedsimilar cytotoxicities under the conditions of continuous exposure.

FIG. 19 graphically shows that the exemplary HER2-targeting molecule115111 (SEQ ID NO:29) is more potently cytotoxic than other exemplaryHER2-targeting molecules for shorter exposure durations. The percentviability of HER2 positive HCC1954 cells was plotted over the logarithmto base 10 of the administered HER2-targeting molecule concentrations.The top graph of FIG. 19 shows that the exemplary HER2-targetingmolecule 115111 (SEQ ID NO:29) was more cytotoxic to HCC1954 cells than114898 (SEQ ID NO:31) under the conditions with 4-hour exposures. Themiddle graph of FIG. 19 shows that the exemplary HER2-targetingmolecules 115111 (SEQ ID NO:29) and 115195 (SEQ ID NO:32) exhibitedsimilar cytotoxicities to each other under the conditions of both 4-hourexposures and continuous exposure and that these molecules exhibited theleast difference in cytotoxic potency when comparing the 4 hour (short)incubation results with the continuous exposure results. The bottomgraph of FIG. 19 shows that the exemplary HER2-targeting molecule 115645(SEQ ID NO:34) and 115845 (SEQ ID NO:35) exhibited similarcytotoxicities to each other under the conditions of both 4-hourexposures and continuous exposure and that the activity of both of theseHER2-targeting molecules was reduced in cytotoxic potency under theshorter four hour incubation with the HER2-targeting molecule ascompared to continuous exposure for days.

FIG. 20 graphically shows the in vitro HER2 binding characteristics ofexemplary HER2-targeting molecules of the present invention usingrecombinant HER2 proteins of human (SEQ ID NO:39), mouse (SEQ ID NO:42),or cynomolgus monkey (SEQ ID NO:40) origin. The top section of FIG. 20graphs the ELISA signal for 114912 (SEQ ID NO:28), 115111 (SEQ IDNO:29), and 115195 (SEQ ID NO:32) tested over a series of HER2-targetingmolecule concentrations. The background subtracted ELISA signal measuredin absorbance at 450 nanometers (nm) is graphed on the Y-axis versus theHER2-targeting molecule concentration in ng/mL on the x-axis. The 115111(SEQ ID NO:29) and 115195 (SEQ ID NO:32) bound both human HER2 ECDprotein (“huHER2”) and cynomolgus monkey HER2 ECD protein (“cynoHER2”)with similar binding characteristics, which appeared to be at slightlyhigher affinities at most concentrations in this assay than the HER2binding exhibited by 114912 (SEQ ID NO:28), a trastuzumab bindingdomain-derived molecule. The bottom section of FIG. 20 shows thebackground subtracted ELISA signal measured in absorbance at 450 nm forthe binding of 115111 (SEQ ID NO:29) to human HER2, cynomolgus monkeyHER2, or mouse HER2 tested at 10 µg/mL of HER2-targeting molecule. Theexemplary HER2-targeting molecule 115111 (SEQ ID NO:29) bound bothrecombinant human HER2 protein and recombinant cynomolgus monkey HERprotein but did not exhibit appreciable binding to recombinant mouseHER2 protein in this assay.

FIG. 21 graphically shows the body weight of immunocompetent miceadministered repeat doses of exemplary HER2-targeting molecules of thepresent invention. The mean body weight change per treatment groupcalculated using the pre-dose weights of the mice in each group aregraphed on the Y-axis versus the day of the study. Groups of BALB/c micewere intravenously administered a vehicle-only control or 1 milligramper kilogram (mg/kg) of body weight of one of these exemplaryHER2-targeting molecules: 115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23),115195 (SEQ ID NO:32), or 115194 (SEQ ID NO:33). In the 11594 (SEQ IDNO:33) treatment group, all mice had died by study Day 12. In the 115195(SEQ ID NO:32) treatment group, all but one of the mice died by studyDay 14. By contrast, the 115111 (SEQ ID NO:29) treatment group showedminimal weight changes similar to the vehicle only control group.

FIG. 22 graphically shows the body weight of immunocompetent miceadministered repeat doses of exemplary HER2-targeting molecules of thepresent invention. The mean body weight change per treatment groupcalculated using the pre-dose weights of the mice in each group aregraphed on the Y-axis versus the day of the study. Groups of C57BL/6mice were intravenously administered a vehicle-only control or 1 mg/kgof body weight of one of these exemplary HER2-targeting molecules:115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), or 115411 (SEQ ID NO:30).In the 115172 (SEQ ID NO:23) treatment group, all mice had died by studyDay 23. By contrast, the mice in the 115111 (SEQ ID NO:29) treatmentgroup tolerated 115111 (SEQ ID NO:29) dosing through study Day 45 (endof the study).

FIG. 23 shows that administration of the exemplary HER2-targetingmolecule 115111 (SEQ ID NO:29) comprising a de-immunized Shiga toxineffector polypeptide resulted in reduced, in vivo, antibody response(s)by a mammalian immune system compared to 115172 (SEQ ID NO:23), whichcomprised a less de-immunized Shiga toxin effector polypeptide. BALB/cmice were administered either 115111 (SEQ ID NO:29) or 115172 (SEQ IDNO:23) at doses between 0.25 to 1 mg/kg body weight. The top graph ofFIG. 23 shows the amount of anti-drug antibodies measured in the bloodsera of the 115111 (SEQ ID NO:29) treatment group as a percentage of the115172 (SEQ ID NO:23) treatment group measured during different days ofa study using BALB/c mice administered 0.25 mg/kg body weight of 115111(SEQ ID NO:29) or 115172 (SEQ ID NO:23) by intraperitoneal injection(IP). The bottom graph of FIG. 23 shows the ELISA signal measured asabsorbance at 450 nm shows the amount of anti-drug antibodies measuredin blood sera collected on study Day 22 of a study using groups ofBALB/c mice intravenously (IV) administered 1 mg/kg body weight of115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), or a vehicle-only control.The sera from the 115111 (SEQ ID NO:29) treatment group exhibited muchless anti-drug antibodies than the sera from 115172 (SEQ ID NO:23)treatment group collected on Day 22.

FIG. 24 graphically shows the results from a subcutaneous HCC1954xenograft murine model study of human breast cancer. The top section ofFIG. 24 graphs the change in human tumor burdens over time for groups ofSCID Beige mice after receiving either the exemplary HER2-targetingmolecule 115111 (SEQ ID NO:29) of the present invention or avehicle-only control sample. The mean tumor volume measured in cubicmillimeters for each group of mice was graphed versus time (dayspost-tumor implant). Administration of the exemplary HER2-targetingmolecule 115111 (SEQ ID NO:29) delayed and reduced the increase in tumorburden observed for the vehicle only control group at all dosagesdisplayed, 0.1 mg to 2 mg per kilogram body weight per dose in cyclesover 31 to 33 days. The bottom section of FIG. 24 graphs the survival ofgroups of mice in the same study as above until Day 84 using a KaplanMeier estimator plot. On the y-axis is the percent survival of micewithin a dosage group, and the x-axis is in days of the study. Therepeated administration of 115111 (SEQ ID NO:29) at 0.1 to 2 mg/kg bodyweight provided survival benefits compared to the vehicle-only controlsample.

DETAILED DESCRIPTION

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art.

In order that the present invention may be more readily understood,certain terms are defined below. Additional definitions may be foundwithin the detailed description of the invention.

As used in the specification and the appended claims, the terms “a,”“an” and “the” include both singular and the plural referents unless thecontext clearly dictates otherwise.

As used in the specification and the appended claims, the term “and/or”when referring to two species, A and B, means at least one of A and B.As used in the specification and the appended claims, the term “and/or”when referring to greater than two species, such as A, B, and C, meansat least one of A, B, or C, or at least one of any combination of A, B,or C (with each species in singular or multiple possibility).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout this specification, the term “including” is used to mean“including but not limited to”. “Including” and “including but notlimited to” are used interchangeably.

As used herein, the term “a plurality of” means more than one; such asat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, or 23.

The term “amino acid residue” or “amino acid” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.The term “polypeptide” includes any polymer of amino acids or amino acidresidues. The term “polypeptide sequence” refers to a series of aminoacids or amino acid residues which physically comprise a polypeptide. A“protein” is a macromolecule comprising one or more polypeptides orpolypeptide “chains.” A “peptide” is a small polypeptide of sizes lessthan about a total of 15 to 20 amino acid residues. The term “amino acidsequence” refers to a series of amino acids or amino acid residues whichphysically comprise a peptide or polypeptide depending on the length.Unless otherwise indicated, polypeptide and protein sequences disclosedherein are written from left to right representing their order from anamino-terminus to a carboxy-terminus.

The terms “amino acid,” “amino acid residue,” “amino acid sequence,” orpolypeptide sequence include naturally occurring amino acids (includingL and D isosteriomers) and, unless otherwise limited, also include knownanalogs of natural amino acids that can function in a similar manner asnaturally occurring amino acids, such as selenocysteine, pyrrolysine,N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine,pyroglutamic acid, and selenomethionine. The amino acids referred toherein are described by shorthand designations as follows in Table A:

TABLE A. Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp DCysteine Cys C Glutamic Acid or Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The phrase “conservative substitution” with regard to an amino acidresidue of a peptide, peptide region, polypeptide region, protein, ormolecule refers to a change in the amino acid composition of thepeptide, peptide region, polypeptide region, protein, or molecule thatdoes not substantially alter the function and structure of the overallpeptide, peptide region, polypeptide region, protein, or molecule (seeCreighton, Proteins: Structures and Molecular Properties (W. H. Freemanand Company, New York (2nd ed., 1992))).

As used herein, the term “HER2” is used interchangeably with the terms“neu” and “ErbB2”.

For purposes of the present invention, the phrase “derived from” whenreferring to a polypeptide or polypeptide region means that thepolypeptide or polypeptide region comprises amino acid sequencesoriginally found in a “parental” protein and which may now comprisecertain amino acid residue additions, deletions, truncations,rearrangements, or other alterations relative to the originalpolypeptide or polypeptide region as long as a certain function(s) and astructure(s) of the “parental” molecule are substantially conserved. Theskilled worker will be able to identify a parental molecule from which apolypeptide or polypeptide region was derived using techniques known inthe art, e.g., protein sequence alignment software.

For purposes of the claimed invention and with regard to a Shiga toxinpolypeptide sequence or Shiga toxin derived polypeptide, the term“wild-type” generally refers to a naturally occurring, Shiga toxinprotein sequence(s) found in a living species, such as, e.g., apathogenic bacterium, wherein that Shiga toxin protein sequence(s) isone of the most frequently occurring variants. This is in contrast toinfrequently occurring Shiga toxin protein sequences that, while stillnaturally occurring, are found in less than one percent of individualorganisms of a given species when sampling a statistically powerfulnumber of naturally occurring individual organisms of that species whichcomprise at least one Shiga toxin protein variant. A clonal expansion ofa natural isolate outside its natural environment (regardless of whetherthe isolate is an organism or molecule comprising biological sequenceinformation) does not alter the naturally occurring requirement as longas the clonal expansion does not introduce new sequence variety notpresent in naturally occurring populations of that species and/or doesnot change the relative proportions of sequence variants to each other.

The terms “associated,” “associating,” “linked,” or “linking” withregard to the claimed invention refers to the state of two or morecomponents of a molecule being joined, attached, connected, or otherwisecoupled to form a single molecule or the act of making two moleculesassociated with each other to form a single molecule by creating anassociation, linkage, attachment, and/or any other connection betweenthe two molecules. For example, the term “linked” may refer to two ormore components associated by one or more atomic interactions such thata single molecule is formed and wherein the atomic interactions may becovalent and/or non-covalent. Non-limiting examples of covalentassociations between two components include peptide bonds andcysteine-cysteine disulfide bonds. Non-limiting examples of non-covalentassociations between two molecular components include ionic bonds.

For purposes of the present invention, the term “linked” refer to two ormore molecular components associated by one or more atomic interactionssuch that a single molecule is formed and wherein the atomicinteractions includes at least one covalent bond. For purposes of thepresent invention, the term “linking” refers to the act of creating alinked molecule as described above.

For purposes of the present invention, the term “fused” refers to two ormore proteinaceous components associated by at least one covalent bondwhich is a peptide bond, regardless of whether the peptide bond involvesthe participation of a carbon atom of a carboxyl acid group or involvesanother carbon atom, such as, e.g., the α-carbon, β-carbon, γ-carbon,σ-carbon, etc. Non-limiting examples of two proteinaceous componentsfused together include, e.g., an amino acid, peptide, or polypeptidefused to a polypeptide via a peptide bond such that the resultingmolecule is a single, continuous polypeptide. For purposes of thepresent invention, the term “fusing” refers to the act of creating afused molecule as described above, such as, e.g., a fusion proteingenerated from the recombinant fusion of genetic regions which whentranslated produces a single proteinaceous molecule.

The symbol “::” means the polypeptide regions before and after it arephysically linked together to form a continuous polypeptide.

As used herein, the terms “expressed,” “expressing,” or “expresses,” andgrammatical variants thereof, refer to translation of a polynucleotideor nucleic acid into a protein. The expressed protein may remainintracellular, become a component of the cell surface membrane or besecreted into an extracellular space.

As used herein, cells which express a significant amount of anextracellular target biomolecule at least one cellular surface are“target positive cells” or “target+ cells” and are cells physicallycoupled to the specified, extracellular target biomolecule.

As used herein, the symbol “α” is shorthand for an immunoglobulin-typebinding region capable of binding to the biomolecule following thesymbol. The symbol “α” is used to refer to the functional characteristicof an immunoglobulin-type binding region based on its ability to bind tothe biomolecule following the symbol with a binding affinity describedby a dissociation constant (K_(D)) of 10⁻⁵ or less.

As used herein, the term “heavy chain variable (V_(H)) domain” or “lightchain variable (V_(L)) domain” respectively refer to any antibody V_(H)or V_(L) domain (e.g. a human V_(H) or V_(L) domain) as well as anyderivative thereof retaining at least qualitative antigen bindingability of the corresponding native antibody (e.g. a humanized V_(H) orV_(L) domain derived from a native murine V_(H) or V_(L) domain). AV_(H) or V_(L) domain consists of a “framework” region interrupted bythe three CDRs or ABRs. The framework regions serve to align the CDRs orABRs for specific binding to an epitope of an antigen. Fromamino-terminus to carboxy-terminus, both V_(H) and V_(L) domainscomprise the following framework (FR) and CDR regions or ABR regions:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2,ABR2, FR3, ABR3, and FR4. As used herein, the terms “HCDR1,” “HCDR2,” or“HCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V_(H)domain, and the terms “LCDR1,” “LCDR2,” and “LCDR3” are used to refer toCDRs 1, 2, or 3, respectively, in a V_(L) domain. As used herein, theterms “HABR1,” “HABR2,” or “HABR3” are used to refer to ABRs 1, 2, or 3,respectively, in a V_(H) domain, and the terms “LABR1,” “LABR2,” or“LABR3” are used to refer to ABRs 1, 2, or 3, respectively, in a V_(L)domain. For camelid V_(H)H fragments, IgNARs of cartilaginous fish,V_(NAR) fragments, certain single domain antibodies, and derivativesthereof, there is a single, heavy chain variable domain comprising thesame basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Asused herein, the terms “HCDR1,” “HCDR2,” or “HCDR3” may be used to referto CDRs 1, 2, or 3, respectively, in a single heavy chain variabledomain.

For purposes of the present invention, the term “effector” meansproviding a biological activity, such as cytotoxicity, biologicalsignaling, enzymatic catalysis, subcellular routing, and/orintermolecular binding resulting in an allosteric effect(s) and/or therecruitment of one or more factors.

For purposes of the present invention, the phrases “Shiga toxin ASubunit effector polypeptide”, “Shiga toxin effector polypeptide,”“Shiga toxin effector polypeptide region,” and “Shiga toxin effectorregion” refer to a polypeptide or polypeptide region derived from atleast one Shiga toxin A Subunit of a member of the Shiga toxin familywherein the polypeptide or polypeptide region is capable of exhibitingat least one Shiga toxin function. For example, SEQ ID NOs: 19-21 arederived from StxA and SLT-1A.

For purposes of the present invention, a Shiga toxin effector functionis a biological activity conferred by a polypeptide region derived froma Shiga toxin A Subunit or an original Shiga toxin A Subunit.Non-limiting examples of Shiga toxin effector functions includepromoting cell entry; lipid membrane deformation; promoting cellularinternalization; stimulating clathrin-mediated endocytosis; directingintracellular routing to various intracellular compartments such as,e.g., the Golgi, endoplasmic reticulum, and cytosol; directingintracellular routing with a cargo; inhibiting a ribosome function(s);catalytic activities, such as, e.g., N-glycosidase activity andcatalytically inhibiting ribosomes; reducing protein synthesis, inducingcaspase activity, activating effector caspases, effectuating cytostaticeffects, and cytotoxicity. Shiga toxin catalytic activities include, forexample, ribosome inactivation, protein synthesis inhibition,N-glycosidase activity, polynucleotide:adenosine glycosidase activity,RNase activity, and DNase activity. Shiga toxins are ribosomeinactivating proteins (RIPs). RIPs can depurinate nucleic acids,polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (and polyA),and viral nucleic acids (see e.g., Barbieri L et al., Biochem J 286: 1-4(1992); Barbieri L et al., Nature 372: 624 (1994); Ling J et al., FEBSLett 345: 143-6 (1994); Barbieri L et al., Biochem J 319: 507-13(1996);Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20 (1996); Stirpe F etal., FEBS Lett 382: 309-12 (1996); Barbieri L et al., Nucleic Acids Res25: 518-22 (1997); Wang P, Turner N, Nucleic Acids Res 27: 1900-5(1999); Barbieri L et al., Biochim Biophys Acta 1480: 258-66 (2000);Barbieri L et al., J Biochem 128: 883-9 (2000); Brigotti M et al.,Toxicon 39: 341-8 (2001); Brigotti M et al., FASEB J 16: 365-72 (2002);Bagga S et al., J Biol Chem 278: 4813-20 (2003); Picard D et al., J BiolChem 280:20069-75 (2005)). Some RIPs show antiviral activity andsuperoxide dismutase activity (Erice A et al., Antimicrob AgentsChemother 37: 835-8 (1993); Au T et al., FEBS Lett 471: 169-72 (2000);Parikh B, Turner N, Mini Rev Med Chem 4: 523-43 (2004); Sharma N et al.,Plant Physiol 134: 171-81 (2004)). Shiga toxin catalytic activities havebeen observed both in vitro and in vivo. Non-limiting examples of assaysfor Shiga toxin effector activity measure various activities, such as,e.g., protein synthesis inhibitory activity, depurination activity,inhibition of cell growth, cytotoxicity, supercoiled DNA relaxationactivity, and nuclease activity.

As used herein, the retention of Shiga toxin effector function refers tobeing capable of exhibiting a level of Shiga toxin functional activity,as measured by an appropriate quantitative assay with reproducibility,comparable to a wild-type, Shiga toxin effector polypeptide control(e.g. a Shiga toxin A1 fragment) or cell-targeting molecule comprising awild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1fragment) under the same conditions. For the Shiga toxin effectorfunction of ribosome inactivation or ribosome inhibition, retained Shigatoxin effector function is exhibiting an IC₅₀ of 10,000 pM or less in anin vitro setting, such as, e.g., by using an assay known to the skilledworker and/or described herein. For the Shiga toxin effector function ofcytotoxicity in a target positive cell-kill assay, retained Shiga toxineffector function is exhibiting a CD₅₀ of 1,000 nM or less, depending onthe cell type and its expression of the appropriate extracellular targetbiomolecule, as shown, e.g., by using an assay known to the skilledworker and/or described herein.

For purposes of the claimed invention, the term “equivalent” with regardto ribosome inhibition means an empirically measured level of ribosomeinhibitory activity, as measured by an appropriate quantitative assaywith reproducibility, which is reproducibly within 10% or less of theactivity of the reference molecule (e.g., the second cell-targetingmolecule, third cell-targeting molecule, etc.) under the sameconditions.

For purposes of the claimed invention, the term “equivalent” with regardto cytotoxicity means an empirically measured level of cytotoxicity, asmeasured by an appropriate quantitative assay with reproducibility,which is reproducibly within 10% or less of the activity of thereference molecule (e.g., the second cell-targeting molecule, thirdcell-targeting molecule, etc.) under the same conditions.

As used herein, the term “attenuated” with regard to cytotoxicity meansa molecule exhibits or exhibited a CD₅₀ between 10-fold to 100-fold of aCD₅₀ exhibited by a reference molecule under the same conditions.

Inaccurate IC₅₀ and CD₅₀ values should not be considered whendetermining a level of Shiga toxin effector function activity. For somesamples, accurate values for either IC₅₀ or CD₅₀ might be unobtainabledue to the inability to collect the required data points for an accuratecurve fit. For example, theoretically, neither an IC₅₀ nor CD₅₀ can bedetermined if greater than 50% ribosome inhibition or cell death,respectively, does not occur in a concentration series for a givensample. Data insufficient to accurately fit a curve as described in theanalysis of the data from exemplary Shiga toxin effector functionassays, such as, e.g., assays described in the Examples below, shouldnot be considered as representative of actual Shiga toxin effectorfunction.

A failure to detect activity in Shiga toxin effector function may be dueto improper expression, polypeptide folding, and/or protein stabilityrather than a lack of cell entry, subcellular routing, and/or enzymaticactivity. Assays for Shiga toxin effector functions may not require muchpolypeptide of the invention to measure significant amounts of Shigatoxin effector function activity. To the extent that an underlying causeof low or no effector function is determined empirically to relate toprotein expression or stability, one of skill in the art may be able tocompensate for such factors using protein chemistry and molecularengineering techniques known in the art, such that a Shiga toxinfunctional effector activity may be restored and measured. As examples,improper cell-based expression may be compensated for by using differentexpression control sequences; and improper polypeptide folding and/orstability may benefit from stabilizing terminal sequences, orcompensatory mutations in non-effector regions which stabilize thethree-dimensional structure of the molecule.

Certain Shiga toxin effector functions are not easily measurable, e.g.subcellular routing functions. For example, there is no routine,quantitative assay to distinguish whether the failure of a Shiga toxineffector polypeptide to be cytotoxic and/or deliver a heterologousepitope is due to improper subcellular routing, but at a time when testsare available, then Shiga toxin effector polypeptides may be analyzedfor any significant level of subcellular routing as compared to theappropriate wild-type Shiga toxin effector polypeptide. However, if aShiga toxin effector polypeptide component of a cell-targeting moleculeof the present invention exhibits cytotoxicity comparable or equivalentto a wild-type Shiga toxin A Subunit construct, then the subcellularrouting activity level is inferred to be comparable or equivalent,respectively, to the subcellular routing activity level of a wild-typeShiga toxin A Subunit construct at least under the conditions tested.

When new assays for individual Shiga toxin functions become available,Shiga toxin effector polypeptides and/or cell-targeting moleculescomprising Shiga toxin effector polypeptides may be analyzed for anylevel of those Shiga toxin effector functions, such as a being within1000-fold or 100-fold or less the activity of a wild-type Shiga toxineffector polypeptide or exhibiting 3-fold to 30-fold or greater activityas compared to a functional knockout, Shiga toxin effector polypeptide.

Sufficient subcellular routing may be merely deduced by observing amolecule’s cytotoxic activity levels in cytotoxicity assays, such as,e.g., cytotoxicity assays based on T-cell epitope presentation or basedon a toxin effector function involving a cytosolic and/or endoplasmicreticulum-localized, target substrate.

As used herein, the retention of “significant” Shiga toxin effectorfunction refers to a level of Shiga toxin functional activity, asmeasured by an appropriate quantitative assay with reproducibilitycomparable to a wild-type Shiga toxin effector polypeptide control (e.g.a Shiga toxin A1 fragment). For in vitro ribosome inhibition,significant Shiga toxin effector function is exhibiting an IC₅₀ of 300pM or less depending on the source of the ribosomes used in the assay(e.g. a bacterial, archaeal, or eukaryotic (algal, fungal, plant, oranimal) source). This is significantly greater inhibition as compared tothe approximate IC₅₀ of 100,000 pM for the catalytically disruptedSLT-1A 1-251 double mutant (Y77S/E167D). For cytotoxicity in atarget-positive cell-kill assay in laboratory cell culture, significantShiga toxin effector function is exhibiting a CD₅₀ of 100, 50, 30 nM, orless, depending on the target biomolecule(s) of the binding region andthe cell type, particularly that cell type’s expression and/orcell-surface representation of the appropriate extracellular targetbiomolecule(s) and/or the extracellular epitope(s) targeted by themolecule being evaluated . This is significantly greater cytotoxicity tothe appropriate, target-positive cell population as compared to a Shigatoxin A Subunit alone (or a wild-type Shiga toxin A1 fragment), withouta cell targeting binding region, which has a CD₅₀ of 100-10,000 nM,depending on the cell line.

For purposes of the present invention and with regard to the Shiga toxineffector function of a molecule of the present invention, the term“reasonable activity” refers to exhibiting at least a moderate level(e.g. within 11-fold to 1,000-fold) of Shiga toxin effector activity asdefined herein in relation to a molecule comprising a naturallyoccurring (or wild-type) Shiga toxin, wherein the Shiga toxin effectoractivity is selected from the group consisting of: internalizationefficiency, subcellular routing efficiency to the cytosol, deliveredepitope presentation by a target cell(s), ribosome inhibition, andcytotoxicity. For cytotoxicity, a reasonable level of Shiga toxineffector activity includes being within 1,000-fold of a wild-type, Shigatoxin construct, such as, e.g., exhibiting a CD₅₀ of 500 nM or less whena wild-type Shiga toxin construct exhibits a CD₅₀ of 0.5 nM (e.g. acell-targeting molecule comprising a wild-type Shiga toxin A1 fragment).

For purposes of the present invention and with regard to thecytotoxicity of a molecule of the present invention, the term “optimal”refers to a level of Shiga toxin catalytic domain mediated cytotoxicitythat is within 2, 3, 4, 5, 6, 7, 8, 9, or 10 -fold of the cytotoxicityof a molecule comprising wild-type Shiga toxin A1 fragment (e.g. a Shigatoxin A Subunit or certain truncated variants thereof) and/or anaturally occurring Shiga toxin.

It should be noted that even if the cytotoxicity of a Shiga toxineffector polypeptide is reduced relative to a wild-type Shiga toxin ASubunit or fragment thereof, in practice, applications using attenuated,Shiga toxin effector polypeptides may be equally or more effective thanusing wild-type Shiga toxin effector polypeptides because the highestpotency variants might exhibit undesirable effects which are minimizedor reduced in reduced cytotoxic-potency variants. Wild-type Shiga toxinsare very potent, being able to kill an intoxicated cell after only onetoxin molecule has reached the cytosol of the intoxicated cell orperhaps after only forty toxin molecules have been internalized into theintoxicated cell. Shiga toxin effector polypeptides with evenconsiderably reduced Shiga toxin effector functions, such as, e.g.,subcellular routing or cytotoxicity, as compared to wild-type Shigatoxin effector polypeptides may still be potent enough for practicalapplications, such as, e.g., applications involving targetedcell-killing, heterologous epitope delivery, and/or detection ofspecific cells and their subcellular compartments. In addition, certainreduced-activity Shiga toxin effector polypeptides may be particularlyuseful for delivering cargos (e.g. an additional exogenous material orT-cell epitope) to certain intracellular locations or subcellularcompartments of target cells.

The term “selective cytotoxicity” with regard to the cytotoxic activityof a molecule refers to the relative level of cytotoxicity between abiomolecule target positive cell population (e.g. a targeted cell-type)and a non-targeted bystander cell population (e.g. a biomolecule targetnegative cell-type), which can be expressed as a ratio of thehalf-maximal cytotoxic concentration (CD₅₀) for a targeted cell typeover the CD₅₀ for an untargeted cell type to provide a metric ofcytotoxic selectivity or indication of the preferentiality of killing ofa targeted cell versus an untargeted cell.

The cell surface representation and/or density of a given extracellulartarget biomolecule (or extracellular epitope of a given targetbiomolecule) may influence the applications for which certaincell-targeting molecules of the present invention may be most suitablyused. Differences in cell surface representation and/or density of agiven target biomolecule between cells may alter, both quantitativelyand qualitatively, the efficiency of cellular internalization and/orcytotoxicity potency of a given cell-targeting molecule of the presentinvention. The cell surface representation and/or density of a giventarget biomolecule can vary greatly among target biomolecule positivecells or even on the same cell at different points in the cell cycle orcell differentiation. The total cell surface representation of a giventarget biomolecule and/or of certain extracellular epitopes of a giventarget biomolecule on a particular cell or population of cells may bedetermined using methods known to the skilled worker, such as methodsinvolving fluorescence-activated cell sorting (FACS) flow cytometry.

As used herein, the terms “disrupted,” “disruption,” or “disrupting,”and grammatical variants thereof, with regard to a polypeptide region orfeature within a polypeptide refers to an alteration of at least oneamino acid within the region or composing the disrupted feature. Aminoacid alterations include various mutations, such as, e.g., a deletion(such as a truncation), inversion, insertion, or substitution whichalter the amino acid sequence of the polypeptide. Amino acid alterationsalso include chemical changes, such as, e.g., the alteration one or moreatoms in an amino acid functional group or the addition of one or moreatoms to an amino acid functional group.

As used herein, “de-immunized” means reduced antigenic and/orimmunogenic potential after administration to a chordate as compared toa reference molecule, such as, e.g., a wild-type peptide region,polypeptide region, or polypeptide. This includes a reduction in overallantigenic and/or immunogenic potential despite the introduction of oneor more, de novo, antigenic and/or immunogenic epitopes as compared to areference molecule. For certain embodiments, “de-immunized” means amolecule exhibited reduced antigenicity and/or immunogenicity afteradministration to a mammal as compared to a “parental” molecule fromwhich it was derived, such as, e.g., a wild-type Shiga toxin A1fragment. In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention is capable of exhibiting a relativeantigenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than the antigenicityof the reference molecule under the same conditions measured by the sameassay, such as, e.g., an assay known to the skilled worker and/ordescribed herein like a quantitative ELISA or Western blot analysis. Incertain embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention is capable of exhibiting a relativeimmunogenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater thanthe immunogenicity of the reference molecule under the same conditionsmeasured by the same assay, such as, e.g., an assay known to the skilledworker and/or described herein like a quantitative measurement ofanti-molecule antibodies produced in a mammal(s) after receivingparenteral administration of the molecule at a given time-point.

The relative immunogenicities of exemplary cell-targeting molecules weredetermined using an assay for in vivo antibody responses to thecell-targeting molecules after repeat, parenteral administrations overperiods of time.

For purposes of the present invention, the phrase “B-cell and/or CD4+T-cell de-immunized” means that the molecule has a reduced antigenicand/or immunogenic potential after administration to a mammal regardingeither B-cell antigenicity or immunogenicity and/or CD4+ T-cellantigenicity or immunogenicity. For certain embodiments, “B-cellde-immunized” means a molecule exhibited reduced B-cell antigenicityand/or immunogenicity after administration to a mammal as compared to a“parental” molecule from which it was derived, such as, e.g., awild-type Shiga toxin A1 fragment. For certain embodiments, “CD4+ T-cellde-immunized” means a molecule exhibited reduced CD4 T-cell antigenicityand/or immunogenicity after administration to a mammal as compared to a“parental” molecule from which it was derived, such as, e.g., awild-type Shiga toxin A1 fragment.

The term “endogenous” with regard to a B-cell epitope, CD4+ T-cellepitope, B-cell epitope region, or CD4+ T-cell epitope region in a Shigatoxin effector polypeptide refers to an epitope present in a wild-typeShiga toxin A Subunit.

For purposes of the present invention, the phrase “CD8+ T-cellhyper-immunized” means that the molecule, when present inside anucleated, chordate cell within a living chordate, has an increasedantigenic and/or immunogenic potential regarding CD8+ T-cellantigenicity or immunogenicity. Commonly, CD8+ T-cell immunizedmolecules are capable of cellular internalization to an early endosomalcompartment of a nucleated, chordate cell due either to an inherentfeature(s) or as a component of a cell-targeting molecule.

For purposes of the present invention, the term “heterologous” means ofa different source than an A Subunit of a naturally occurring Shigatoxin, e.g. a heterologous polypeptide is not naturally found as part ofany A Subunit of a native Shiga toxin. The term “heterologous” withregard to T-cell epitope or T-cell epitope-peptide component of apolypeptide of the present invention refers to an epitope or peptidesequence which did not initially occur in the polypeptide to bemodified, but which has been added to the polypeptide, whether added viathe processes of embedding, fusion, insertion, and/or amino acidsubstitution as described herein, or by any other engineering means. Theresult is a modified polypeptide comprising a T-cell epitope foreign tothe original, unmodified polypeptide, i.e. the T-cell epitope was notpresent in the original polypeptide.

The term “embedded” and grammatical variants thereof with regard to aT-cell epitope or T-cell epitope-peptide component of a polypeptide ofthe present invention refers to the internal replacement of one or moreamino acids within a polypeptide region with different amino acids inorder to generate a new polypeptide sequence sharing the same totalnumber of amino acid residues with the starting polypeptide region.Thus, the term “embedded” does not include any external, terminal fusionof any additional amino acid, peptide, or polypeptide component to thestarting polypeptide nor any additional internal insertion of anyadditional amino acid residues, but rather includes only substitutionsfor existing amino acids. The internal replacement may be accomplishedmerely by amino acid residue substitution or by a series ofsubstitutions, deletions, insertions, and/or inversions. If an insertionof one or more amino acids is used, then the equivalent number ofproximal amino acids must be deleted next to the insertion to result inan embedded T-cell epitope. This is in contrast to use of the term“inserted” with regard to a T-cell epitope contained within apolypeptide of the present invention to refer to the insertion of one ormore amino acids internally within a polypeptide resulting in a newpolypeptide having an increased number of amino acid residues comparedto the starting polypeptide.

The term “inserted” and grammatical variants thereof with regard to aT-cell epitope contained within a polypeptide of the present inventionrefers to the insertion of one or more amino acids within a polypeptideresulting in a new polypeptide sequence having an increased number ofamino acid residues compared to the starting polypeptide. The “pure”insertion of a T-cell epitope-peptide is when the resulting polypeptideincreased in length by the number of amino acid residues equivalent tothe number of amino acid residues in the entire, inserted T-cellepitope-peptide. The phrases “partially inserted,” “embedded andinserted,” and grammatical variants thereof with regard to a T-cellepitope contained within a polypeptide of the present invention, refersto when the resulting polypeptide increased in length, but by less thanthe number of amino acid residues equivalent to the length of theentire, inserted T-cell epitope-peptide. Insertions, whether “pure” or“partial,” include any of the previously described insertions even ifother regions of the polypeptide not proximal to the insertion sitewithin the polypeptide are deleted thereby resulting in a decrease inthe total length of the final polypeptide because the final polypeptidestill comprises an internal insertion of one or more amino acids of aT-cell epitope-peptide within a polypeptide region.

As used herein, the term “T-cell epitope delivering” when describing afunctional activity of a molecule means that a molecule provides thebiological activity of localizing within a cell to a subcellularcompartment that is competent to result in the proteasomal cleavage of aproteinaceous part of the molecule which comprises a T-cellepitope-peptide. The “T-cell epitope delivering” function of a moleculecan be assayed by observing the MHC presentation of a T-cellepitope-peptide cargo of the molecule on a cell surface of a cellexogenously administered the molecule or in which the assay was begunwith the cell containing the molecule in one or more of its endosomalcompartments. Generally, the ability of a molecule to deliver a T-cellepitope to a proteasome can be determined where the initial location ofthe “T-cell epitope delivering” molecule is an early endosomalcompartment of a cell, and then, the molecule is empirically shown todeliver the epitope-peptide to the proteasome of the cell. However, a“T-cell epitope delivering” ability may also be determined where themolecule starts at an extracellular location and is empirically shown,either directly or indirectly, to deliver the epitope into a cell and toproteasomes of the cell. For example, certain “T-cell epitope delivering“molecules pass through an endosomal compartment of the cell, such as,e.g. after endocytic entry into that cell. Alternatively, “T-cellepitope delivering” activity may be observed for a molecule starting atan extracellular location whereby the molecule does not enter anyendosomal compartment of a cell-instead the “T-cell epitope delivering”molecule enters a cell and delivers a T-cell epitope-peptide toproteasomes of the cell, presumably because the “T-cell epitopedelivering” molecule directed its own routing to a subcellularcompartment competent to result in proteasomal cleavage of its T-cellepitope-peptide component.

For purposes of the present invention, the phrase “proximal to anamino-terminus” with reference to the position of a Shiga toxin effectorpolypeptide region of a cell-targeting molecule of the present inventionrefers to a distance wherein at least one amino acid residue of theShiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 or more, e.g., up to 18-20 amino acid residues, of anamino-terminus of the cell-targeting molecule as long as thecell-targeting molecule is capable of exhibiting the appropriate levelof Shiga toxin effector functional activity noted herein (e.g., acertain level of cytotoxic potency). Thus for certain embodiments of thepresent invention, any amino acid residue(s) fused amino-terminal to theShiga toxin effector polypeptide should not reduce any Shiga toxineffector function (e.g., by sterically hindering a structure(s) near theamino-terminus of the Shiga toxin effector polypeptide region) such thata functional activity of the Shiga toxin effector polypeptide is reducedbelow the appropriate activity level required herein.

For purposes of the present invention, the phrase “more proximal to anamino-terminus” with reference to the position of a Shiga toxin effectorpolypeptide region within a cell-targeting molecule of the presentinvention as compared to another component (e.g., a cell-targeting,binding region, molecular moiety, and/or additional exogenous material)refers to a position wherein at least one amino acid residue of theamino-terminus of the Shiga toxin effector polypeptide is closer to theamino-terminus of a linear, polypeptide component of the cell-targetingmolecule of the present invention as compared to the other referencedcomponent.

For purposes of the present invention, the phrase “active enzymaticdomain derived from one A Subunit of a member of the Shiga toxin family”refers to having the ability to inhibit protein synthesis via acatalytic ribosome inactivation mechanism. The enzymatic activities ofnaturally occurring (or wild-type) Shiga toxins may be defined by theability to inhibit protein translation using assays known to the skilledworker, such as, e.g., in vitro assays involving RNA translation in theabsence of living cells or in vivo assays involving RNA translation in aliving cell. Using assays known to the skilled worker and/or describedherein, the potency of a Shiga toxin enzymatic activity may be assesseddirectly by observing N-glycosidase activity toward ribosomal RNA(rRNA), such as, e.g., a ribosome nicking assay, and/or indirectly byobserving inhibition of ribosome function and/or protein synthesis.

For purposes of the present invention, the term “Shiga toxin A1 fragmentregion” refers to a polypeptide region consisting essentially of a Shigatoxin A1 fragment and/or derived from a Shiga toxin A1 fragment of aShiga toxin.

For purposes of the present invention, the terms “terminus,”“amino-terminus,” or “carboxy-terminus” with regard to a cell-targetingmolecule refers generally to the last amino acid residue of apolypeptide chain of the cell-targeting molecule (e.g., a single,continuous polypeptide chain). A cell-targeting molecule may comprisemore than one polypeptides or proteins, and, thus, a cell-targetingmolecule of the present invention may comprise multiple amino-terminalsand carboxy-terminals. For example, the “amino-terminus” of acell-targeting molecule may be defined by the first amino acid residueof a polypeptide chain representing the amino-terminal end of thepolypeptide, which is generally characterized by a starting, amino acidresidue which does not have a peptide bond with any amino acid residueinvolving the primary amino group of the starting amino acid residue orinvolving the equivalent nitrogen for starting amino acid residues whichare members of the class of N-alkylated alpha amino acid residues.Similarly, the “carboxy-terminus” of a cell-targeting molecule may bedefined by the last amino acid residue of a polypeptide chainrepresenting the carboxyl-terminal end of the polypeptide, which isgenerally characterized by a final, amino acid residue which does nothave any amino acid residue linked by a peptide bond to the alpha-carbonof its primary carboxyl group.

For purposes of the present invention, the terms “terminus,”“amino-terminus,” or “carboxy-terminus” with regard to a polypeptideregion refers to the regional boundaries of that region, regardless ofwhether additional amino acid residues are linked by peptide bondsoutside of that region. In other words, the terminals of the polypeptideregion regardless of whether that region is fused to other peptides orpolypeptides. For example, a fusion protein comprising two proteinaceousregions, e.g., a binding region comprising a peptide or polypeptide anda Shiga toxin effector polypeptide, may have a Shiga toxin effectorpolypeptide region with a carboxy-terminus ending at amino acid residue251 of the Shiga toxin effector polypeptide region despite a peptidebond involving residue 251 to an amino acid residue at position 252representing the beginning of another proteinaceous region, e.g., thebinding region. In this example, the carboxy-terminus of the Shiga toxineffector polypeptide region refers to residue 251, which is not aterminus of the fusion protein but rather represents an internal,regional boundary. Thus, for polypeptide regions, the terms “terminus,”“amino-terminus,” and “carboxy-terminus” are used to refer to theboundaries of polypeptide regions, whether the boundary is a physicallyterminus or an internal, position embedded within a larger polypeptidechain.

For purposes of the present invention, the phrase “carboxy-terminusregion of a Shiga toxin A1 fragment” refers to a polypeptide regionderived from a naturally occurring (or wild-type) Shiga toxin A1fragment, the region beginning with a hydrophobic residue (e.g., V236 ofStxA-A1 and SLT-1A1, and V235 of SLT-2A1) that is followed by ahydrophobic residue and the region ending with the furin-cleavage siteconserved among Shiga toxin A1 fragment polypeptides and ending at thejunction between the A1 fragment and the A2 fragment in native, Shigatoxin A Subunits. For purposes of the present invention, thecarboxy-terminal region of a Shiga toxin A1 fragment includes a peptidicregion derived from the carboxy-terminus of a Shiga toxin A1 fragmentpolypeptide, such as, e.g., a peptidic region comprising, consistingessentially of, or consisting of the carboxy-terminus of a Shiga toxinA1 fragment. Non-limiting examples of peptidic regions derived from thecarboxy-terminus of a Shiga toxin A1 fragment include the amino acidresidue sequences natively positioned from position 236 to position 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251 in Stx1A(SEQ ID NO:2) or SLT-1A (SEQ ID NO: 1); and from position 235 toposition 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250in SLT-2A (SEQ ID NO:3).

For purposes of the present invention, the phrase “proximal to thecarboxy-terminus of an A1 fragment polypeptide” with regard to a linkedmolecular moiety and/or binding region refers to being within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues from the amino acidresidue defining the last residue of the Shiga toxin A1 fragmentpolypeptide.

For purposes of the present invention, the phrase “sterically covers thecarboxy-terminus of the A1 fragment-derived region” includes anymolecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) linked and/or fused to an amino acidresidue in the carboxy-terminus of the A1 fragment-derived region, suchas, e.g., the amino acid residue derived from the amino acid residuenatively positioned at any one of positions 236 to 251 in StxlA (SEQ IDNO:2) or SLT-1A (SEQ ID NO: 1) or from 235 to 250 in SLT-2A (SEQ IDNO:3). For purposes of the present invention, the phrase “stericallycovers the carboxy-terminus of the A1 fragment-derived region” alsoincludes any molecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) linked and/or fused to an amino acidresidue in the carboxy-terminus of the A1 fragment-derived region, suchas, e.g., the amino acid residue carboxy-terminal to the last amino acidA1 fragment-derived region and/or the Shiga toxin effector polypeptide.For purposes of the present invention, the phrase “sterically covers thecarboxy-terminus of the A1 fragment-derived region” also includes anymolecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) physically preventing cellularrecognition of the carboxy-terminus of the A1 fragment-derived region,such as, e.g. recognition by the ERAD machinery of a eukaryotic cell.

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin binding region or an immunoglobulin-type bindingregion, that comprises a polypeptide comprising at least forty aminoacids and that is linked (e.g., fused) to the carboxy-terminus of theShiga toxin effector polypeptide region comprising an A1fragment-derived region is a molecular moiety which is “stericallycovering the carboxy-terminus of the A1 fragment-derived region.”

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin binding region or an immunoglobulin-type bindingregion, that comprises a polypeptide comprising at least forty aminoacids and that is linked (e.g., fused) to the carboxy-terminus of theShiga toxin effector polypeptide region comprising an A1fragment-derived region is a molecular moiety “encumbering thecarboxy-terminus of the A1 fragment-derived region.”

For purposes of the present invention, the term “A1 fragment of a memberof the Shiga toxin family” refers to the remaining amino-terminalfragment of a Shiga toxin A Subunit after proteolysis by furin at thefurin-cleavage site conserved among Shiga toxin A Subunits andpositioned between the A1 fragment and the A2 fragment in wild-typeShiga toxin A Subunits.

For purposes of the claimed invention, the phrase “furin-cleavage motifat the carboxy-terminus of the A1 fragment region” refers to a specific,furin-cleavage motif conserved among Shiga toxin A Subunits and bridgingthe junction between the A1 fragment and the A2 fragment in naturallyoccurring, Shiga toxin A Subunits.

For purposes of the present invention, the phrase “furin-cleavage siteproximal to the carboxy-terminus of the A1 fragment region” refers toany identifiable, furin-cleavage site having an amino acid residuewithin a distance of less than 1, 2, 3, 4, 5, 6, 7 or more amino acidresidues of the amino acid residue defining the last amino acid residuein the A1 fragment region or A1 fragment derived region, including afurin-cleavage motif located carboxy-terminal of an A1 fragment regionor A1 fragment derived region, such as, e.g., at a position proximal tothe linkage of the A1 fragment-derived region to another component ofthe molecule, such as, e.g., a molecular moiety of a cell-targetingmolecule of the present invention.

For purposes of the present invention, the phrase “disruptedfurin-cleavage motif” refers to (i) a specific furin-cleavage motif asdescribed herein in Section I-B and (ii) which comprises a mutationand/or truncation that can confer a molecule with a reduction infurin-cleavage as compared to a reference molecule, such as, e.g., areduction in furin-cleavage reproducibly observed to be 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for nocleavage) than the furin-cleavage of a reference molecule observed inthe same assay under the same conditions. The percentage offurin-cleavage as compared to a reference molecule can be expressed as aratio of cleaved:uncleaved material of the molecule of interest dividedby the cleaved:uncleaved material of the reference molecule (see e.g. WO2015/191764; WO 2016/196344). Non-limiting examples of suitablereference molecules include certain molecules comprising a wild-typeShiga toxin furin-cleavage motif and/or furin-cleavage site as describedherein and/or molecules used as reference molecules in the Examplesbelow.

For purposes of the present invention, the phrase “furin-cleavageresistant” means a molecule or specific polypeptide region thereofexhibits reproducibly less furin cleavage than (i) the carboxy-terminusof a Shiga toxin A1 fragment in a wild-type Shiga toxin A Subunit or(ii) the carboxy-terminus of the Shiga toxin A1 fragment derived regionof construct wherein the naturally occurring furin-cleavage sitenatively positioned at the junction between the A1 and A2 fragments isnot disrupted; as assayed by any available means to the skilled worker,including by using a method described herein.

For purposes of the present invention, the phrase “active enzymaticdomain derived form an A Subunit of a member of the Shiga toxin family”refers to a polypeptide structure having the ability to inhibit proteinsynthesis via catalytic inactivation of a ribosome based on a Shigatoxin enzymatic activity. The ability of a molecular structure toexhibit inhibitory activity of protein synthesis and/or catalyticinactivation of a ribosome may be observed using various assays known tothe skilled worker, such as, e.g., in vitro assays involving RNAtranslation assays in the absence of living cells or in vivo assaysinvolving the ribosomes of living cells. For example, using assays knownto the skilled worker, the enzymatic activity of a molecule based on aShiga toxin enzymatic activity may be assessed directly by observingN-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., aribosome nicking assay, and/or indirectly by observing inhibition ofribosome function, RNA translation, and/or protein synthesis.

As used herein with respect to a Shiga toxin effector polypeptide, a“combination” describes a Shiga toxin effector polypeptide comprisingtwo or more sub-regions wherein each sub-region comprises at least oneof the following: (1) a disruption in an endogenous epitope or epitoperegion; (2) an embedded, heterologous, T-cell epitope-peptide; (3) aninserted, heterologous, T-cell epitope-peptide; and (4) a disruptedfurin-cleavage motif at the carboxy-terminus of an A1 fragment region.

As used herein, the term “additional HER2-targeting therapeutic agent”means an additional therapeutic agent (e.g. a molecule) that targetsHER2 to produce a therapeutic effect or benefit. This additionalHER2-targeting therapeutic agent is complementary to the cell-targetingmolecule of the present invention and does not compete directly with thecell-targeting molecule in its HER2-targeting activity. The additionalHER2-targeting therapeutic agent may comprise, consist essentially of,or consist of an anti-HER2 antibody or small molecule inhibitor thatinterferes with HER2 signaling. For example, the additionalHER2-targeting therapeutic agent may comprise, consist essentially of,or consists of a dual tyrosine kinase inhibitor, such as lapatiniband/or neratinib. The additional HER2-targeting therapeutic agent maycomprise, consist essentially of, or consist of an anti-HER2 antibodytherapy that binds to an antigenic determinant that does not overlapwith the antigenic determinant bound by the cell-targeting molecule ofthe invention or that binds a HER2 molecule in such a manner that whenbound the additional HER2-tageting therapeutic does not prevent thebinding of that HER2 molecule by the cell-targeting molecule of theinvention. For example, the additional HER2-targeting therapeutic agentmay comprise, consist essentially of, or consist of anti-HER2 monoclonalantibody therapy and/or anti-HER2 antibody drug conjugate therapy, suchas, e.g., T-DM1 (trastuzumab emtansine), trastuzumab, and/ orpertuzumab. The additional HER2-targeting therapeutic agent may beselected from any one of or a combination of: lapatinib, neratinib,T-DM1 (trastuzumab emtansine), trastuzumab, and/or pertuzumab.

As used herein with respect to a molecule of the present invention, a“cell-targeting molecule” is used interchangeably with a “HER2-targetingmolecule” or “HER2-binding molecule”. All of the aforementioned moleculetypes include various “HER2-binding proteins”.

INTRODUCTION

The present invention provides various cell-targeting moleculescomprising one or more Shiga toxin effector polypeptides and at leastone HER2-binding region. Certain embodiments of the cell-targetingmolecules of the present invention comprise Shiga toxin effectorpolypeptides that combine structural elements resulting in two or moreproperties in a single molecule, such as, e.g., the ability to 1)exhibit reduced antigenicity and/or immunogenicity as compared tomolecular variants lacking that particular combination of elements, 2)exhibit reduced protease-cleavage as compared to molecular variantslacking that particular combination of elements, 3) exhibit reducednon-specific toxicity to a multicellular organism at certain dosages ascompared to molecular variants lacking that particular combination ofelements, and/or 5) exhibit potent cytotoxicity. The cell-targetingmolecules of the present invention may serve as scaffolds to createvarious cell-targeting molecules, such as, e.g., HER2-targeted,cytotoxic, therapeutic molecules; HER2-targeted, nontoxic, deliveryvehicles; and HER2-targeted diagnostic molecules.

I. The General Structures of the Cell-Targeting Molecules of the PresentInvention

The present invention provides various cell-targeting molecules, eachcomprising (1) a cell-targeting, binding region and (2) a Shiga toxineffector polypeptide component. The Shiga toxin effector polypeptides ofthe present invention may be associated with and/or coupled to various,diverse, cell-targeting components (e.g. a molecular moiety and/oragent) to create cell-targeting molecules of the present invention. Acell-targeting molecule of the present invention comprises (1) a bindingregion capable of specifically binding an extracellular part of a targetbiomolecule and (2) a Shiga toxin effector polypeptide capable ofexhibiting one or more Shiga toxin A Subunit effector functions. Theassociation of a cell-targeting binding region(s) with a Shiga toxineffector polypeptide of the present invention enables the engineering oftherapeutic and diagnostic molecules with desirable characteristics,such as, e.g., de-immunization, potent cytotoxicity, efficientintracellular routing, T-cell hyper-immunization, molecular stability,and in vivo tolerability at high dosages as compared to certainreference molecules.

The present invention provides various HER2-targeting molecules, eachcomprising (1) a cell-targeting, binding region capable of binding HER2and (2) a Shiga toxin A Subunit effector polypeptide capable ofexhibiting a Shiga toxin effector function. The Shiga toxin effectorpolypeptide may be associated with and/or coupled to various, diverse,HER2-targeting components (e.g. a molecular moiety and/or agent) tocreate cell-targeting molecules of the present invention. Acell-targeting molecule of the present invention comprises (1) a bindingregion capable of specifically binding an extracellular part of a HER2target biomolecule and (2) a Shiga toxin effector polypeptide regioncomprising a Shiga toxin effector polypeptide capable of exhibiting oneor more Shiga toxin A Subunit effector functions, such as, e.g.,cytostasis, cytotoxicity, catalytic activity, promoting cellularinternalization, directing intracellular routing to a certainsubcellular compartment(s), and intracellular delivery of a material(s).For example, the cell-targeting molecules of the present invention maycomprise a Shiga toxin A Subunit effector polypeptide component thatcomprises a Shiga toxin A1 fragment derived region, wherein the Shigatoxin A Subunit effector polypeptide comprises: (a) an embedded orinserted, heterologous, CD8+ T-cell epitope which disrupts anendogenous, B-cell and/or CD4+ T-cell epitope region; and (b) adisruption of at least three, endogenous, B-cell and/or CD4+ T-cellepitope regions which do not overlap with the embedded or inserted,heterologous, CD8+ T-cell epitope; wherein the Shiga toxin effectorpolypeptide comprises a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment region, wherein saidfurin-cleavage motif is disrupted by a carboxy-terminal truncation ofthe Shiga toxin A1 fragment region as compared to the carboxy-terminusof a wild-type Shiga toxin A Subunit; wherein the Shiga toxin A subuniteffector polypeptide is capable of exhibiting a Shiga toxin effectorfunction.

The Shiga toxin effector polypeptides of the present invention may belinked to one or more cell-targeting, binding regions that mediatecell-targeting via binding specificity to extracellular parts of targetbiomolecules, such as, e.g., a HER2 target biomolecule physicallycoupled to a cellular surface of a cell. One non-limiting example of acell-targeting molecule of the present invention is a Shiga toxineffector polypeptide of the present invention fused to a proteinaceous,cell-targeting, binding region, such as, e.g., an immunoglobulin orimmunoglobulin-type binding region. For example, the cell-targetingmolecules of the present invention may comprise an immunoglobulinbinding region capable of specifically binding an extracellular part ofHER2/neu/ErbB2, and comprising a polypeptide comprising one or more of:an antibody variable fragment, a single-domain antibody fragment, asingle-chain variable fragment, a Fd fragment, an antigen-bindingfragment, an autonomous VH domain, a V_(H)H fragment derived from acamelid antibody, a heavy-chain antibody domain derived from acartilaginous fish antibody, a VNAR fragment, and an immunoglobulin newantigen receptor.

A. HER2/Neu/ErbB2 Binding Regions

In certain embodiments, a binding region of a cell-targeting molecule ofthe present invention is a cell-targeting component, such as, e.g., adomain, molecular moiety, or agent, capable of binding specifically toan extracellular part of a HER2/neu/ErbB2 target biomolecule on a cellsurface (i.e. an extracellular target biomolecule) with high affinity.There are numerous types of binding regions known to skilled worker orwhich may be discovered by the skilled worker using techniques known inthe art. For example, any cell-targeting component that exhibits therequisite binding characteristics described herein may be used as thebinding region in certain embodiments of the cell-targeting molecules ofthe present invention.

An extracellular part of a target biomolecule refers to a portion of itsstructure exposed to the extracellular environment when the molecule isphysically coupled to a cell, such as, e.g., when the target biomoleculeis expressed at a cellular surface by the cell. In this context, exposedto the extracellular environment means that part of the targetbiomolecule is accessible by, e.g., an antibody or at least a bindingmoiety smaller than an antibody such as a single-domain antibody domain,a nanobody®, a heavy-chain antibody domain derived from camelids orcartilaginous fishes, a single-chain variable fragment, or any number ofengineered alternative scaffolds to immunoglobulins (see below). Theexposure to the extracellular environment of or accessibility to a partof target biomolecule physically coupled to a cell may be empiricallydetermined by the skilled worker using methods well known in the art.

HER2, also recognized in the art as receptor tyrosine-protein kinaseerbB-2, is a transmembrane protein which functions as a cell surfacereceptor for transducing signals across the cellular membrane tointracellular regulators of cell proliferation and apoptosis. HER2 isalso recognized in the art as Neu, erbB-2, p185, CD340, NGL, andHER2/neu (Coussens L et al., Science 230: 1132-39 (1985); King C et al.,Science 229: 974-6 (1985); Semba K et al., Proc Natl Acad Sci USA 82:6497-501 (1985); Yamamoto T et al., Nature 319:230-234 (1986); Kokai Yet al., Proc Natl Acad Sci USA 85: 5389-93 (1988); Disis M et al.,Cancer Res 54: 16-20 (1994); Yoshino I et al., J Immunol 152: 2393-400(1994) see e.g., GenBank Acc. Nos. X03363; M17730; NM_004448; SEG_HUMHER20). While the name HER2 might refer to multiple proteins withrelated structures and polypeptide sequences from various species, forthe purposes of the structural examples of this section, the term “HER2”refers to the epidermal growth factor receptor proteins present inhumans whose exact sequence might vary slightly based on the isoform andfrom individual to individual. For example, HER2 refers to the humanprotein represented by the exemplary polypeptide sequences UniProtP04626 and NCBI accessions NP_004439.2, NP_(_)001005862.1,NP_001276865.1, NP_001276866.1, and NP_001276867.1; however, differentisoforms and variants exist due to splicing, polymorphisms and/ormutations (see e.g. Siddig A et al., Ann NYAcad Sci 1138: 84-94 (2008);Poole E et al., IntJMol Epidemiol Genet 2: 300-15 (2011); WO2000/020579). A skilled worker will be able to identify other HER2proteins in humans, even if they differ from the referenced sequences.

HER2 is overexpressed by many cancer cells, notably breast cancer cells,and its overexpression is strongly associated with increased metastasis,increased disease reoccurrence, and poor prognosis (see e.g. Slamon D etal., Science 235: 177-82 (1987)).

There are numerous HER2 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “HER2 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a HER2molecule with high affinity, such as, e.g., having a dissociationconstant with regard to HER2 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, HER2 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human HER2 (also known as neu orErbB2).

A binding region of a cell-targeting molecule of the present inventionmay be, e.g., a ligand, peptide, immunoglobulin-type binding region,monoclonal antibody, engineered antibody derivative, or engineeredalternative to antibodies. For example, the binding region is animmunoglobulin binding region.

In certain embodiments, the binding region of a cell-targeting moleculeof the present invention is a proteinaceous moiety capable of bindingspecifically to an extracellular part of target biomolecule with highaffinity. A binding region of a cell-targeting molecule of the presentinvention may comprise one or more various peptidic or polypeptidemoieties, such as randomly generated peptide sequences, naturallyoccurring ligands or derivatives thereof, immunoglobulin deriveddomains, synthetically engineered scaffolds as alternatives toimmunoglobulin domains, and the like (see e.g., WO 2005/092917; WO2007/033497; Cheung M et al., Mol Cancer 9: 28 (2010); US 2013/0196928;WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO2015/191764). In certain embodiments, a cell-targeting molecule of thepresent invention comprises a binding region comprising one or morepolypeptides capable of selectively and specifically binding anextracellular target biomolecule.

There are numerous binding regions known in the art that are useful fortargeting molecules to extracellular portions of HER2 via their bindingcharacteristics, such as certain monoclonal antibodies, engineeredantibody derivatives, and engineered alternatives to antibodies.

According to one specific, but non-limiting aspect, the binding regionmay comprise an immunoglobulin-type binding region. The term“immunoglobulin-type binding region” as used herein refers to apolypeptide region capable of binding one or more target biomolecules,such as an antigen or epitope. Binding regions may be functionallydefined by their ability to bind to target molecules.Immunoglobulin-type binding regions are commonly derived from antibodyor antibody-like structures; however, alternative scaffolds from othersources are contemplated within the scope of the term. In certainembodiments, the binding region may comprise an immunoglobulin bindingregion derived from antibody or antibody-like structure.

Immunoglobulin (Ig) proteins have a structural domain known as an Igdomain. Ig domains range in length from about 70-110 amino acid residuesand possess a characteristic Ig-fold, in which typically 7 to 9antiparallel beta strands arrange into two beta sheets which form asandwich-like structure. The Ig fold is stabilized by hydrophobic aminoacid interactions on inner surfaces of the sandwich and highly conserveddisulfide bonds between cysteine residues in the strands. Ig domains maybe variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgIor I-set). Some Ig domains may be associated with a complementaritydetermining region (CDR), also called a “complementary determiningregion,” which is important for the specificity of antibodies binding totheir epitopes. Ig-like domains are also found in non-immunoglobulinproteins and are classified on that basis as members of the Igsuperfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC)provides a list of members of the Ig-like domain containing family.

An immunoglobulin-type binding region may be a polypeptide sequence ofan antibody or antigen-binding fragment thereof wherein the amino acidsequence has been varied from that of a native antibody or an Ig-likedomain of a non-immunoglobulin protein, for example by molecularengineering or selection by library screening. Because of the relevanceof recombinant DNA techniques and in vitro library screening in thegeneration of immunoglobulin-type binding regions, antibodies can beredesigned to obtain desired characteristics, such as smaller size, cellentry, or other improvements for in vivo and/or therapeuticapplications. The possible variations are many and may range from thechanging of just one amino acid to the complete redesign of, forexample, a variable region. Typically, changes in the variable regionwill be made in order to improve the antigen-binding characteristics,improve variable region stability, or reduce the potential forimmunogenic responses.

There are numerous immunoglobulin-type binding regions contemplated ascomponents of the present invention. In certain embodiments, theimmunoglobulin-type binding region is derived from an immunoglobulinbinding region, such as an antibody paratope capable of binding anextracellular target biomolecule. In certain other embodiments, theimmunoglobulin-type binding region comprises an engineered polypeptidenot derived from any immunoglobulin domain but which functions like animmunoglobulin binding region by providing high-affinity binding to anextracellular target biomolecule. This engineered polypeptide mayoptionally include polypeptide scaffolds comprising, consisting of, orconsisting essentially of complementary determining regions fromimmunoglobulins as described herein.

There are also numerous binding regions in the prior art that are usefulfor targeting polypeptides to specific cell-types via theirhigh-affinity binding characteristics. In certain embodiments, thebinding region of the cell-targeting molecule of the present inventionis selected from the group which includes autonomous V_(H) domains,single-domain antibody domains (sdAbs), heavy-chain antibody domainsderived from camelids (V_(H)H fragments or V_(H) domain fragments),heavy-chain antibody domains derived from camelid V_(H)H fragments orV_(H) domain fragments, heavy-chain antibody domains derived fromcartilaginous fishes, immunoglobulin new antigen receptors (IgNARs),V_(NAR) fragments, single-chain variable (scFv) fragments, nanobodies®,Fd fragments consisting of the heavy chain and C_(H)1 domains, singlechain Fv-CH3 minibodies, dimeric C_(H)2 domain fragments (C_(H)2D), Fcantigen binding domains (Fcabs), isolated complementary determiningregion 3 (CDR3) fragments, constrained framework region 3, CDR3,framework region 4 (FR3-CDR3-FR4) polypeptides, small modularimmunopharmaceutical (SMIP) domains, scFv-Fc fusions, multimerizing scFvfragments (diabodies, triabodies, tetrabodies), disulfide stabilizedantibody variable (Fv) fragments, disulfide stabilized antigen-binding(Fab) fragments consisting of the V_(L), V_(H), C_(L) and C_(H)1domains, bivalent nanobodies®, bivalent minibodies, bivalent F(ab′)₂fragments (Fab dimers), bispecific tandem V_(H)H fragments, bispecifictandem scFv fragments, bispecific nanobodies®, bispecific minibodies,and any genetically manipulated counterparts of the foregoing thatretain its paratope and binding function (see Ward E et al., Nature 341:544-6 (1989); Davies J, Riechmann L, Biotechnology (NY) 13: 475-9(1995); Reiter Y et al., Mol Biol 290: 685-98 (1999); Riechmann L,Muyldermans S, J ImmunolMethods 231: 25-38 (1999); Tanha J et al., JImmunol Methods 263: 97-109 (2002); Vranken W et al., Biochemistry 41:8570-9 (2002); Jespers L et al., J Mol Biol 337: 893-903 (2004); JespersL et al., NatBiotechnol 22: 1161-5 (2004); To R et al., J Biol Chem 280:41395-403 (2005); Saerens D et al., Curr Opin Pharmacol 8: 600-8 (2008);Dimitrov D, MAbs 1: 26-8 (2009); Weiner L, Cell 148: 1081-4 (2012);Ahmad Z et al., Clin Dev Immunol 2012: 980250 (2012)). For example, thecell-targeting molecule of the present invention may comprise a bindingregion that comprises, consists essentially of, or consists of one ormore of: an antibody variable fragment, a single-domain antibodyfragment, a single-chain variable fragment, a Fd fragment, anantigen-binding fragment, an autonomous VH domain, a V_(H)H fragmentderived from a camelid antibody, a heavy-chain antibody domain derivedfrom a cartilaginous fish antibody, a VNAR fragment, and animmunoglobulin new antigen receptor. In certain further embodiments, thebinding region comprises, consists essentially of, or consists of asingle-chain variable fragment and/or a V_(H)H fragment derived from acamelid antibody. In certain further embodiments, the binding regioncomprises, consists essentially of, or consists of a single-chainvariable fragment. In certain further embodiments, the binding regioncomprises, consists essentially of, or consists of a V_(H)H fragmentderived from a camelid antibody.

There are a variety of binding regions comprising polypeptides derivedfrom the constant regions of immunoglobulins, such as, e.g., engineereddimeric Fc domains, monomeric Fcs (mFcs), scFv-Fcs, V_(H)H-Fcs, C_(H)2domains, monomeric C_(H)3s domains (mC_(H)3s), syntheticallyreprogrammed immunoglobulin domains, and/or hybrid fusions ofimmunoglobulin domains with ligands (Hofer T et al., Proc Natl Acad SciU. S. A. 105: 12451-6 (2008); Xiao J et al., J Am Chem Soc 131:13616-13618 (2009); Xiao X et al., Biochem Biophys Res Commun 387:387-92 (2009); Wozniak-Knopp G et al., Protein Eng Des Sel 23 289-97(2010); Gong R et al., PLoS ONE 7: e42288 (2012); Wozniak-Knopp G etal., PLoS ONE 7: e30083 (2012); Ying T et al., JBiol Chem 287: 19399-408(2012); Ying T et al., J Biol Chem 288: 25154-64 (2013); Chiang M etal., J Am Chem Soc 136: 3370-3 (2014); Rader C, Trends Biotechnol 32:186-97 (2014); Ying T et al., Biochimica Biophys Acta 1844: 1977-82(2014)).

In accordance with certain other embodiments, the binding regioncomprises an engineered, alternative scaffold to immunoglobulin domains.Engineered alternative scaffolds are known in the art which exhibitsimilar functional characteristics to immunoglobulin-derived structures,such as high-affinity and specific binding of target biomolecules, andmay provide improved characteristics to certain immunoglobulin domains,such as, e.g., greater stability or reduced immunogenicity. Generally,alternative scaffolds to immunoglobulins are less than 20 kilodaltons,consist of a single polypeptide chain, lack cysteine residues, andexhibit relatively high thermodynamic stability.

In certain embodiments of the cell-targeting molecules of the presentinvention, the binding region comprises an alternative scaffold selectedfrom the group which includes autonomous V_(H) domains, single-domainantibody domains (sdAbs), heavy-chain antibody domains derived fromcamelids (V_(H)H fragments or V_(H) domain fragments), heavy-chainantibody domains derived from camelid V_(H)H fragments or V_(H) domainfragments, heavy-chain antibody domains derived from cartilaginousfishes, immunoglobulin new antigen receptors (IgNARs), V_(NAR)fragments, single-chain variable (scFv) fragments, nanobodies®, Fdfragments consisting of the heavy chain and C_(H)1 domains, permutatedFvs (pFv), single chain Fv-CH3 minibodies, dimeric C_(H)2 domainfragments (C_(H)2D), Fc antigen binding domains (Fcabs), isolatedcomplementary determining region 3 (CDR3) fragments, constrainedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4)polypeptides, small modular immunopharmaceutical (SMIP) domains, scFv-Fcfusions, multimerizing scFv fragments (diabodies, triabodies,tetrabodies), disulfide stabilized antibody variable (Fv) fragments,disulfide stabilized antigen-binding (Fab) fragments consisting of theV_(L), V_(H), C_(L) and C_(H)1 domains, bivalent nanobodies®, bivalentminibodies, bivalent F(ab′)₂ fragments (Fab dimers), bispecific tandemV_(H)H fragments, bispecific tandem scFv fragments, bispecificnanobodies®, bispecific minibodies, and any genetically manipulatedcounterparts of the foregoing that retains its binding functionality(Wörn A, Plückthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., ChemBiol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62(2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005); Hey T et al.,Trends Biotechnol 23 :514-522 (2005); Holliger P, Hudson P, NatBiotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17:653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Byla P et al., JBiol Chem 285: 12096 (2010); Zoller F et al., Molecules16: 2467-85 (2011); Alfarano P et al., Protein Sci 21: 1298-314 (2012);Madhurantakam C et al., Protein Sci 21: 1015-28 (2012); Varadamsetty Get al., J Mol Biol 424: 68-87 (2012); Reichen C et al., J Struct Biol185: 147-62 (2014)).

For example, numerous alternative scaffolds have been identified whichbind to an extracellular part of the human cell-surface receptor HER2(see e.g. Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004); OrlovaA et al. Cancer Res 66: 4339-8 (2006); Ahlgren S et al., Bioconjug Chem19: 235-43 (2008); Feldwisch J et al., J MolBiol 398: 232-47 (2010);U.S. pat. 5,578,482; 5,856,110; 5,869,445; 5,985,553; 6,333,169;6,987,088; 7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185;7,449,480; 7,560,111; 7,674,460; 7,815,906; 7,879,325; 7,884,194;7,993,650; 8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967;8,652,474; and U.S. Pat. application 2011/0059090). In addition toalternative antibody formats, antibody-like binding abilities may beconferred by non-proteinaceous compounds, such as, e.g., oligomers, RNAmolecules, DNA molecules, carbohydrates, and glycocalyxcalixarenes (seee.g. Sansone F, Casnati A, Chem Soc Rev 42: 4623-39 (2013)) or partiallyproteinaceous compounds, such as, e.g., phenol-formaldehyde cyclicoligomers coupled with peptides and calixarene-peptide compositions (seee.g. U.S. 5,770,380).

In certain embodiments, the HER2 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, HER2 binding region is derived from animmunoglobulin, HER2 binding region, such as an antibody paratopecapable of binding an extracellular part of HER2. In certain otherembodiments, the immunoglobulin-type, HER2 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, HER2 binding region by providinghigh-affinity binding to an extracellular part of HER2. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising,consisting of, or consisting essentially of complementary determiningregions (such as, e.g., a heavy chain variable domain and/or light chainvariable domain) and/or antigen binding regions from immunoglobulins asdescribed herein.

There are numerous HER2 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,HER2 binding regions include HER2-binding monoclonal antibodies andderivatives thereof, such as, e.g., anti-ErbB2, 4D5, 2C4, 7F3, 7C2,mumAb 4D5, chmAb 4D5, (rhu)mAb 4D5, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, huMAb4D5-8, trastuzumab,humanized 520C9, 4D5Fc8, hingeless rhu4D5, non-glycosylated rhu4D5 withmutated cysteine residues, pertuzumab, and humanized 2C4 (Hudziak R etal., Mol Cell Biol 9: 1165-72 (1989); McKenzie S et al., Oncogene4:543-8 (1989); Bacus S et al., Molecular Carcinogenesis 3: 350-62(1990); Hancock M et al., Cancer Res 51: 4575-80 (1991); Maier L et al.,Cancer Res 51: 5361-5369 (1991); Stancovski I et al., Proc Natl Acad SciUSA 88: 8691-5 (1991); Tagliabue E et al., Int J Cancer 47: 933-937(1991); Bacus S et al., Cancer Res 52: 2580-9 (1992); Carter P et al.,Proc Natl Acad Sci USA 89: 4285-89 (1992); Harwerth I et al. J Biol Chem267: 15160-7 (1992); Kasprzyk P et al., Cancer Res 52: 2771-6 (1992);Lewis G et al., Cancer Immunol Immunother 37: 255-63 (1993); Xu F etal., Int J Cancer 53: 401-8 (1993); Arteaga C et al., Cancer Res 54:3758-65 (1994); Shawver L et al., Cancer Res 54: 1367-73 (1994); KlapperL et al. Oncogene 14: 2099-109 (1997); WO 1993/21319; WO 1994/00136; WO1997/00271; WO 1998/77797; US 5,772,997; US 5,783,186; US 5,821,337; US5,840,525; US 6,949,245; and US 7,625,859).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide (e.g. an immunoglobulin polypeptide) selected for specificand high-affinity binding to human HER2 and/or the cellular surface of aHER2 positive cell. In certain embodiments of the cell-targetingmolecule of the present invention, the binding region comprises at leastone heavy chain variable (V_(H)) domain; and/or at least one light chainvariable (V_(L)) domain. As described herein, the at least oneheavy-chain variable domain polypeptide may be linked to the at leastone light-chain variable domain polypeptide by a linker (such as alinker or inter-domain linker described herein). In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a single-domain antibody fragment, such as, e.g., onlya heavy chain variable (V_(H)H) domain (e.g. as derived from a camelidantibody).

The binding region of the cell-targeting molecule of the presentinvention may be defined by reference to its CDRs, such as those definedin SEQ ID NOs: 45-74. In certain embodiments of the cell-targetingmolecule of the present invention, the binding region comprises apolypeptide(s) selected from the group consisting of: a) a heavy chainvariable (VH) domain comprising (i) a HCDR1 comprising or consistingessentially of one of the amino acid sequences as shown in SEQ ID NO:45,SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:63; (ii) a HCDR2 comprising orconsisting essentially of one of the amino acid sequence as shown in SEQID NO:46, SEQ ID NO:52, SEQ ID NO:58, or SEQ ID NO:64; and (iii) a HCDR3comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO: 47, SEQ ID NO:53, SEQ ID NO:59, or SEQ ID NO:65;and/or b) a light chain variable (VL) domain comprising (i) a LCDR1comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, or SEQ ID NO:66;(ii) a LCDR2 comprising or consisting essentially of one of the aminoacid sequence as shown in SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO: 61 orSEQ ID NO:67; and (iii) a LCDR3 comprising or consisting essentially ofone of the amino acid sequence as shown in SEQ ID NO:50, SEQ ID NO:56,SEQ ID NO:62, or SEQ ID NO:68. In certain embodiments, the bindingregion comprises at least one heavy-chain variable domain polypeptidecomprising (i) the HCDR1, HCDR2, and HCDR3 amino acid sequences shown inSEQ ID NOs: 51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (ii) theHCDR1, HCDR2, and HCDR3 amino acid sequences shown in SEQ ID NO:57, SEQID NO:58, and SEQ ID NO:59, respectively; or (iii) the HCDR1, HCDR2, andHCDR3 amino acid sequences shown in SEQ ID NO:63, SEQ ID NO:64, and SEQID NO:65, respectively. In certain embodiments, the binding regioncomprises at least one light-chain variable domain polypeptidecomprising (i) the LCDR1, LCDR2, and LCDR3 amino acid sequences shown inSEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, respectively; (ii) theLCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:60, SEQID NO: 61, and SEQ ID NO:62, respectively; or (iii) the LCDR1, LCDR2,and LCDR3 amino acid sequences shown in SEQ ID NO:66, SEQ ID NO:67, andSEQ ID NO:68, respectively.

In certain embodiments, the binding region comprises at least oneheavy-chain variable domain polypeptide comprising (i) the HCDR1, HCDR2,and HCDR3 amino acid sequences shown in SEQ ID NOs: 51, SEQ ID NO:52,and SEQ ID NO:53, respectively; (ii) the HCDR1, HCDR2, and HCDR3 aminoacid sequences shown in SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59,respectively; or (iii) the HCDR1, HCDR2, and HCDR3 amino acid sequencesshown in SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; andat least one light-chain variable domain polypeptide comprising (i) theLCDR1, LCDR2, and LCDR3 amino acid sequences shown in SEQ ID NO:54, SEQID NO:55, and SEQ ID NO:56, respectively; (ii) the LCDR1, LCDR2, andLCDR3 amino acid sequences shown in SEQ ID NO:60, SEQ ID NO:61, and SEQID NO:62, respectively; or (iii) the LCDR1, LCDR2, and LCDR3 amino acidsequences shown in SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68,respectively. For example, the binding region may comprises at least oneheavy-chain variable domain polypeptide comprising (i) the HCDR1, HCDR2,and HCDR3 amino acid sequences shown in SEQ ID NOs: 51, SEQ ID NO:52,and SEQ ID NO:53, respectively; and at least one light-chain variabledomain polypeptide comprising: (i) the LCDR1, LCDR2, and LCDR3 aminoacid sequences shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56,respectively. For example, the binding region may comprises at least oneheavy-chain variable domain polypeptide comprising (i) the HCDR1, HCDR2,and HCDR3 amino acid sequences shown in SEQ ID NOs: 57, SEQ ID NO:58,and SEQ ID NO:59, respectively; and at least one light-chain variabledomain polypeptide comprising (i) the LCDR1, LCDR2, and LCDR3 amino acidsequences shown in SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:62,respectively. For example, the binding region may comprises at least oneheavy-chain variable domain polypeptide comprising (i) the HCDR1, HCDR2,and HCDR3 amino acid sequences shown in SEQ ID NOs: 63, SEQ ID NO:64,and SEQ ID NO:65, respectively; and at least one light-chain variabledomain polypeptide comprising (i) the LCDR1, LCDR2, and LCDR3 amino acidsequences shown in SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68,respectively. The binding region having these CDRs may be animmunoglobulin binding region comprising a single-chain variablefragment.

In certain embodiments of the cell-targeting molecule of the presentinvention, the binding region comprises a polypeptide(s) selected fromthe group consisting of: a) a heavy chain only variable (V_(H)H) domaincomprising (i) a HCDR1 comprising or consisting essentially of the aminoacid sequences as shown in SEQ ID NO:69 or SEQ ID NO:72; (ii) a HCDR2comprising or consisting essentially of the amino acid sequence as shownin SEQ ID NO:70 or SEQ ID NO:73; and/or (iii) a HCDR3 comprising orconsisting essentially of the amino acid sequence as shown in SEQ IDNO:71 or SEQ ID NO:74. In certain further embodiments, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain only variable (V_(H)H) domain comprising (i) a HCDR1comprising or consisting essentially of the amino acid sequences asshown in SEQ ID NO:69 or SEQ ID NO:72; (ii) a HCDR2 comprising orconsisting essentially of the amino acid sequence as shown in SEQ IDNO:70 or SEQ ID NO:73; and (iii) a HCDR3 comprising or consistingessentially of the amino acid sequence as shown in SEQ ID NO:71 or SEQID NO:74. The binding region having these CDRs may be an immunoglobulinbinding region comprising a heavy chain only variable (V_(H)H) domainderived from a camelid antibody (see e.g. Example 1, infra).

In certain embodiments of the cell-targeting molecule of the presentinvention, the binding region comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence of: amino acids 269 to 501 of SEQID NO:24; amino acids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499of SEQ ID NO: 26 or SEQ ID NO:27; amino acids; amino acids 269-520 ofSEQ ID NO:28; amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30;amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ IDNO:32; amino acids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; amino acids 269 to514 of SEQ ID NO:36; amino acids 268 to 498 of SEQ ID NO:99; amino acids268 to 499 of SEQ ID NO: 100; amino acids 268 to 500 of SEQ ID NO:97;amino acids 268 to 512 of SEQ ID NO:98; amino acids 268 to 518 of SEQ IDNO: 102 or SEQ ID NO: 103; amino acids 268-519 of SEQ ID NO: 101; aminoacids 267 to 384 of SEQ ID NO: 104; amino acids 268 to 498 of SEQ ID NO:105; amino acids 252 to 370 of SEQ ID NO: 106; amino acids 252 to 366 ofSEQ ID NO: 107; and amino acids 268 to 513 of SEQ ID NO: 108. In certainembodiments of the cell-targeting molecule of the present invention, thebinding region comprises, consists essentially of, or consists of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence of: amino acids 269 to 513 of SEQ ID NO:25; amino acids269 to 499 of SEQ ID NO:26; amino acids 269 to 519 of SEQ ID NO:29 orSEQ ID NO:30; amino acids 268 to 386 of SEQ ID NO:31; amino acids 253 to370 of SEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; or aminoacids 269 to 514 of SEQ ID NO:36. In certain embodiments of thecell-targeting molecule of the present invention, the binding regioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to amino acids 269 to 519 ofSEQ ID NO:29 or SEQ ID NO:30.

In certain embodiments of the cell-targeting molecule of the presentinvention, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by any one of the followingpolypeptide sequences: amino acids 269 to 501 of SEQ ID NO:24; aminoacids 269 to 513 of SEQ ID NO:25; amino acids 269 to 499 of SEQ ID NO:26 or SEQ ID NO:27; amino acids; amino acids 269-520 of SEQ ID NO:28;amino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30; amino acids 268to 386 of SEQ ID NO:31; amino acids 269 to 499 of SEQ ID NO:32; aminoacids 269 to 499 of SEQ ID NO:33; amino acids 253 to 370 of SEQ IDNO:34; amino acids 253 to 367 of SEQ ID NO:35; amino acids 269 to 514 ofSEQ ID NO:36 amino acids 268 to 498 of SEQ ID NO:99; amino acids 268 to499 of SEQ ID NO: 100; amino acids 268 to 500 of SEQ ID NO:97; aminoacids 268 to 512 of SEQ ID NO:98; amino acids 268 to 518 of SEQ ID NO:102 or SEQ ID NO: 103; amino acids 268-519 of SEQ ID NO: 101; aminoacids 267 to 384 of SEQ ID NO: 104; amino acids 268 to 498 of SEQ ID NO:105; amino acids 252 to 370 of SEQ ID NO: 106; amino acids 252 to 366 ofSEQ ID NO: 107; and amino acids 268 to 513 of SEQ ID NO: 108. In certainembodiments of the cell-targeting molecule of the present invention, thebinding region comprises, consists essentially of, or consists of thepolypeptide represented by any one of the following polypeptidesequences: amino acids 269 to 513 of SEQ ID NO:25; amino acids 269 to499 of SEQ ID NO:26; amino acids 269 to 519 of SEQ ID NO:29 or SEQ IDNO:30; amino acids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370 ofSEQ ID NO:34; amino acids 253 to 367 of SEQ ID NO:35; and amino acids269 to 514 of SEQ ID NO:36. In certain embodiments of the cell-targetingmolecule of the present invention, the binding region comprises,consists essentially of, or consists of the polypeptide represented byamino acids 269 to 519 of SEQ ID NO:29 or SEQ ID NO:30. In certainembodiments of the cell-targeting molecule of the present invention, thebinding region comprises, consists essentially of, or consists of thepolypeptide represented by amino acids 269 to 519 of SEQ ID NO:29, aminoacids 268 to 386 of SEQ ID NO:31; amino acids 253 to 370 of SEQ IDNO:34; or amino acids 253 to 367 of SEQ ID NO:35. In certainembodiments, the binding region comprises, consists essentially of, orconsists of the polypeptide represented by amino acids 269 to 519 of SEQID NO:29. In certain, embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byamino acids 268 to 386 of SEQ ID NO:31. In certain embodiments, thebinding region comprises, consists essentially of, or consists of thepolypeptide represented by amino acids 253 to 370 of SEQ ID NO:34. Incertain embodiments, the binding region comprises, consists essentiallyof, or consists of the polypeptide represented by amino acids 253 to 367of SEQ ID NO:35. In certain embodiments, the binding region comprises,consists essentially of, or consists of the polypeptide represented byamino acids 269 to 514 of SEQ ID NO:36.

In certain embodiments of the cell-targeting molecule of the presentinvention, the binding region comprises at least one heavy chainvariable (V_(H)) domain comprising, consisting essentially of, orconsisting of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence shown in any one of: amino acids253 to 367 of SEQ ID NO:35; amino acids 253 to 370 of SEQ ID NO:34;amino acids 268 to 386 of SEQ ID NO:31; amino acids 269 to 387 of SEQ IDNO: 26, 29, 30 or 36; amino acids 269 to 397 of SEQ ID NO:25; aminoacids 381 to 500 of SEQ ID NO: 24 or 27; and amino acids 401 to 520 ofSEQ ID NO:28. In certain further embodiments, the binding regioncomprises at least one heavy chain variable (V_(H)) domain comprising,consisting essentially of, or consisting of: amino acids 253 to 367 ofSEQ ID NO:35; amino acids 253 to 370 of SEQ ID NO:34; amino acids 268 to386 of SEQ ID NO:31; amino acids 269 to 387 of SEQ ID NO: 26, 29, 30 or36; amino acids 269 to 397 of SEQ ID NO:25; amino acids 381 to 500 ofSEQ ID NO: 24 or 27; and amino acids 401 to 520 of SEQ ID NO:28. Incertain embodiments of the cell-targeting molecule of the presentinvention, the binding region comprises at least one heavy chainvariable (V_(H)) domain comprising, consisting essentially of, orconsisting of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to the amino acid sequence shown in any one of: amino acids269 to 387 of SEQ ID NO: 26, 29, 30 or 36; amino acids 269 to 397 of SEQID NO:25; amino acids 381 to 500 of SEQ ID NO: 24 or 27; and amino acids401 to 520 of SEQ ID NO:28. In certain further embodiments of thecell-targeting molecule of the present invention, the binding regioncomprises at least one light chain variable (V_(L)) domain comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more) identical to the amino acid sequence shown in anyone of: amino acids 269 to 375 of SEQ ID NO: 24, 27, or 28; amino acids393 to 499 of SEQ ID NO:26; amino acids 403 to 513 of SEQ ID NO:25;amino acids 408 to 514 of SEQ ID NO:36; and amino acids 413 to 519 ofSEQ ID NO: 29 or 30. In certain further embodiments of thecell-targeting molecule of the present invention, the binding regioncomprises at least one light chain variable (V_(L)) domain comprising,consisting essentially of, or consisting of: amino acids 269 to 375 ofSEQ ID NO: 24, 27, or 28; amino acids 393 to 499 of SEQ ID NO:26; aminoacids 403 to 513 of SEQ ID NO:25; amino acids 408 to 514 of SEQ IDNO:36; and amino acids 413 to 519 of SEQ ID NO: 29 or 30. Any of heavychain variable domain polypeptides described herein may be used incombination with any of the light chain variable domain polypeptidesdescribed herein.

In certain embodiments, the binding region may comprise: (a) at leastone heavy chain variable (V_(H)) domain comprising, consistingessentially of, or consisting of: amino acids 269 to 387 of SEQ ID NOs:26, 29, 30, or 36; amino acids 269 to 397 of SEQ ID NO:25; amino acids381 to 500 of SEQ ID NO: 24 or 27; amino acids 401 to 522 of SEQ IDNO:36, or amino acids 401 to 520 of SEQ ID NO:28; and (b) at least onelight chain variable (V_(L)) domain comprising, consisting essentiallyof, or consisting of: amino acids 269 to 375 of SEQ ID NO: 24, 27, or28; amino acids 393 to 499 of SEQ ID NO:26; amino acids 403 to 513 ofSEQ ID NO:25; amino acids 408 to 514 of SEQ ID NO:36; and amino acids413 to 519 of SEQ ID NO: 29 or 30. For example, the binding region maycomprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 381to 500 of SEQ ID NO:24; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 375 of SEQ ID NO:24. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 269to 397 of SEQ ID NO:25; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting of:amino acids 403 to 513 of SEQ ID NO:25. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 269to 387 of SEQ ID NO:26; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 393 to 499 of SEQ ID NO:26. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 381to 500 of SEQ ID NO:27; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 375 of SEQ ID NO:27. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 401to 520 of SEQ ID NO:28; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 269 to 375 of SEQ ID NO:28. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 269to 387 of SEQ ID NO:29; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 413 to 519 of SEQ ID NO:29. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 269to 387 of SEQ ID NO:30; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 413 to 519 of SEQ ID NO:30. For example, the binding regionmay comprise (a) at least one heavy chain variable (V_(H)) domaincomprising, consisting essentially of, or consisting of amino acids 269to 387 of SEQ ID NO:36; and (b) at least one light chain variable(V_(L)) domain comprising, consisting essentially of, or consisting ofamino acids 408 to 514 of SEQ ID NO:36.

In certain embodiments, the binding region comprises or consistsessentially of amino acids 269-520 of SEQ ID NO: 102.

In certain embodiments, the binding region comprises the heavy chainvariable domain comprising or consisting essentially of amino acids 269to 387 of SEQ ID NO:26, 29-30, or 36; 269 to 397 of SEQ ID NO:25; 381 to500 of SEQ ID NO:27; or 401 to 522 of SEQ ID NO:36. In certain furtherembodiments, the binding region comprises the light chain variabledomain comprising or consisting essentially of amino acids 269 to 375 ofSEQ ID NO:27; 393 to 499 of SEQ ID NO:26; 403 to 513 of SEQ ID NO:25;408 to 514 of SEQ ID NO:36; 413 to 519 of SEQ ID NO:29 or 30. In certainfurther embodiments, the binding region comprises or consistsessentially of amino acids 269 to 513 of SEQ ID NO:25; 269 to 499 of SEQID NO:26; 269 to 519 of SEQ ID NO:29; 269 to 519 of SEQ ID NO:30; 268 to386 of SEQ ID NO:31; 269 to 499 of SEQ ID NO:32; 269 to 499 of SEQ IDNO:33; 253 to 370 of SEQ ID NO:34; 253 to 367 of SEQ ID NO:35; or 269 to514 of SEQ ID NO:36.

A natural ligand or derivative thereof may be utilized as the HER2binding region for a cell-targeting molecule of the present invention.Native HER2 is known to heterodimerize with other members of the ErbBfamily upon binding ligands such as epidermal growth factors likeepiregulin and heregulin (Moasser M, Oncogene 26: 6469-87 (2007); RieseD, Cullum R, Semin Cell Dev Biol 28: 49-56 (2014); Sollome J et al.,Cell Signal 26: 70-82 (2014)). ErbB ligands which bind members of theErbB family include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF,epiregulin, HER2-68 and HER2-100, heregulins, herstatin, NRG-2, NRG-3,and NRG-4 (Justman Q et al., JBiol Chem 277: 20618-24 (2002);Jhabvala-Romero F., et al., Oncogene 22: 8178-86 (2003)). Examples of anErbB ligand include the heregulins (HRG), such as the prototypeheregulin disclosed in U.S. Pat. 5,641,869 and Marchionni M et al.,Nature 362: 312-8 (1993). Examples of heregulins include heregulin-α,heregulin-β1, heregulin-β2 and heregulin-β3 (Holmes W et al., Science256: 1205-10 (1992); US 5,641,869); neu differentiation factor (NDF)(Peles et al., Cell 69: 205-16 (1992)); acetylcholine receptor-inducingactivity (ARIA) (Falls D et al., Cell 72: 801-15 (1993)); glial growthfactors (GGFs) (Marchionni M et al., Nature 362: 312-8 (1993)); sensoryand motor neuron derived factor (SMDF) (Ho W et al., JBiol Chem 270:14523-32 (1995)); γ-heregulin (Schaefer G et al., Oncogene 15: 1385-94(1997)).

An ErbB ligand according to the present invention may also be asynthetic ErbB ligand. The synthetic ligand may be specific for aparticular ErbB receptor or may recognize particular ErbB receptorcomplexes. An example of a synthetic ligand is the syntheticheregulin/EGF chimera biregulin (Jones J et al., FEBS Lett, 447: 227-31(1999)) and the EGF-like domain fragment HRGβ1177-244. ErbB ligands or apart of an ErbB ligand that interacts with HER2 or a derivative thereofmay be fused to Shiga toxin effector polypeptides of the invention toconstruct HER2-targeting, cell-targeting molecules of the invention thatbind an extracellular part of HER2.

Synthetic peptides which bind an extracellular part of HER2 may beutilized as the binding region for targeting. Many peptides have beendescribed which are capable of binding to HER2 (see e.g. U.S. Pat.5,578,482; 5,856,110; 5,869,445; 5,985,553; 6,333,169; 6,987,088;7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480;7,560,111; 7,674,460; 7,815,906, 7,879,325; 7,884,194; 7,993,650;8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967; 8,652,474; and US2011/0059090).

In certain embodiments, small molecules which bind an extracellular partof HER2 may be utilized as the binding region for targeting. Many smallmolecules have been described which are capable of binding to HER2 suchas tyrosine kinase inhibitors, AZD8931, lapatinib, neratinib (HKI-272),dacomitinib (PF-00299804), afatinib (BIBW 2992) (Barlaam B et al., ACSMed Chem Lett 4: 742-6 (2013); Yu H, Riley G, J Natl Compr Canc Netw 11:161-9 (2013); Roskoski R, Pharmacol Res 87C: 42-59 (2014)). Other smallmolecules which bind to an extracellular part of HER2 may be identifiedusing methods well known to those of skill in the art, such as byderivatizing known EGFR binders like gefitinib, erlotinib, AEE788,AG1478, AG1571 (SU-5271), AP26113, CO-1686, XL647, vandetanib, andBMS-690514 (Kurokawa H, Arteaga C, Clin Cancer Res 7: 4436 s-4442s(2001); Yigitbasi O et al., Cancer Res 64: 7977-84 (2004); Yu H, RileyG, J Natl Compr Canc Netw 11: 161-9 (2013); Roskoski R, Pharmacol Res87C: 42-59 (2014)).

Any of the aforementioned HER2 binding molecules may be suitable for useas a HER2 binding region or modified to create one or more HER2 bindingregions for use in a cell-targeting molecule of the present invention.Any of the above binding region structures may be used as a component ofa molecule of the present invention as long as the binding regioncomponent has a dissociation constant of 10⁻⁵ to 10⁻¹² moles per liter,preferably less than 200 nanomolar (nM), towards an extracellular partof a HER2 molecule.

HER2/Neu/ErbB2 Target Biomolecules Bound by the Binding Regions

In certain embodiments, the binding region of a cell-targeting moleculesof the present invention comprises a proteinaceous region capable ofbinding specifically to an extracellular part of a HER2 biomolecule oran extracellular HER2 biomolecule, preferably which is physicallycoupled to the surface of a cell type of interest, such as, e.g., acancer cell and/or tumor cell.

The term “target biomolecule” refers to a biological molecule, commonlya proteinaceous molecule or a protein modified by post-translationalmodifications, such as glycosylation, that is bound by a binding regionof a cell-targeting molecule of the present invention resulting in thetargeting of the cell-targeting molecule to a specific cell, cell-type,and/or location within a multicellular organism.

For purposes of the present invention, the term “extracellular” withregard to a target biomolecule refers to a biomolecule that has at leasta portion of its structure exposed to the extracellular environment. Theexposure to the extracellular environment of or accessibility to a partof target biomolecule coupled to a cell may be empirically determined bythe skilled worker using methods well known in the art. Non-limitingexamples of extracellular target biomolecules include cell membranecomponents, transmembrane spanning proteins, cell membrane-anchoredbiomolecules, cell-surface-bound biomolecules, and secretedbiomolecules.

With regard to the present invention, the phrase “physically coupled”when used to describe a target biomolecule means covalent and/ornon-covalent intermolecular interactions couple the target biomolecule,or a portion thereof, to the outside of a cell, such as a plurality ofnon-covalent interactions between the target biomolecule and the cellwhere the energy of each single interaction is on the order of at leastabout 1-5 kiloCalories (e.g., electrostatic bonds, hydrogen bonds, ionicbonds, Van der Walls interactions, hydrophobic forces, etc.). Allintegral membrane proteins can be found physically coupled to a cellmembrane, as well as peripheral membrane proteins. For example, anextracellular target biomolecule might comprise a transmembrane spanningregion, a lipid anchor, a glycolipid anchor, and/or be non-covalentlyassociated (e.g. via non-specific hydrophobic interactions and/or lipidbinding interactions) with a factor comprising any one of the foregoing.

Extracellular parts of target biomolecules may include various epitopes,including unmodified polypeptides, polypeptides modified by the additionof biochemical functional groups, and glycolipids (see e.g. US5,091,178; EP2431743).

The binding regions of the cell-targeting molecules of the presentinvention may be designed or selected based on numerous criteria, suchas the cell-type specific expression of their HER2 target, the physicallocalization of their HER2 target biomolecules with regard to specificcell types, and/or the properties of their target HER2 biomolecules. Forexample, certain cell-targeting molecules of the present inventioncomprise binding regions capable of binding a cell-surface HER2 targetbiomolecule that is expressed at a cellular surface exclusively by onlyone cell-type of a species or only one cell-type within a multicellularorganism. It is desirable, but not necessary, that an extracellulartarget HER2 biomolecule be intrinsically internalized or be readilyforced to internalize upon interacting with a cell-targeting molecule ofthe present invention.

Among certain embodiments of the cell-targeting molecules of the presentinvention, the binding region is derived from an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to a HER2antigen on the cell surface of a cancer or tumor cell, where the antigenis restricted in expression to cancer or tumor cells (see Glokler J etal., Molecules 15: 2478-90 (2010); Liu Y et al., Lab Chip 9: 1033-6(2009). In accordance with other embodiments, the binding region isselected for specific and high-affinity binding to an extracellular partof HER2 on the cell surface of a cancer cell, where the HER2 isover-expressed or preferentially expressed by cancer cells as comparedto non-cancer cells.

It will be appreciated by the skilled worker that any desired targetHER2 biomolecule may be used to design or select a suitable bindingregion to be associated and/or coupled with a Shiga toxin effectorpolypeptide to produce a cell-targeting molecule of the presentinvention.

Any of the above binding regions described herein may be used alone orin combination with each individual embodiment of the present invention,including methods of the present invention.

The general structure of the cell-targeting molecules of the presentinvention is modular, in that various, diverse, HER2-targeting bindingregions may be associated with various, Shiga toxin effectorpolypeptides of the present invention to create different,cell-targeting molecules of the present invention which exhibitdifferences in their cell-targeting activities due to differences intheir binding regions. This enables a variety of cell-targetingactivities to be exhibited by different embodiments of thecell-targeting molecules of the present invention such that differentembodiments target different types of cells with Shiga toxin effectorfunctions, such as, e.g., cytostasis, cytotoxicity, and intracellulardelivery of exogenous materials. Furthermore, certain embodiments of thecell-targeting molecules of the present invention exhibit certaincharacteristics due to differences in their respective Shiga toxineffector polypeptide regions, such as, e.g., low antigenicity and/orimmunogenicity when administered to a chordate, resistance toproteolytic cleavage by certain proteases, high stability whenadministered to a multicellular organism, in vivo tolerability at highdosages, ability to deliver a cargo to an intracellular location, and/orability to deliver a T-cell epitope to a MHC class I molecule forpresentation on a cellular surface.

For the purposes of the present invention, the specific order ororientation of the Shiga toxin effector polypeptide region and thecell-targeting, HER2-binding region is not fixed in relation to eachother or within the cell-targeting molecule of the present inventionunless expressly noted. For example, when the cell-targeting molecule ofthe present invention is a fusion protein with an amino-terminal(s) andcarboxy-terminal(s), various arrangements of the components of theinvention may be suitable (see e.g. FIG. 1 ). In certain embodiments ofthe cell-targeting molecules of the present invention, the arrangementof their components in relation to each other or within thecell-targeting molecule are limited as described herein. For example,certain endoplasmic reticulum retention/retrieval signal motifs (seee.g. WO 2015/138435) are commonly positioned on a carboxy-terminus of acell-targeting molecule of the present invention and/or acarboxy-terminus of a protein component of a cell-targeting molecule ofthe present invention.

B. The General Structures of the Shiga Toxin A Subunit EffectorPolypeptides

The cell-targeting molecules of the present invention comprise at leastone, Shiga toxin effector polypeptide derived from wild-type Shiga toxinA Subunits that further comprise one or more structural modifications,such as, e.g., a mutation like a truncation and/or amino acid residuesubstitution(s). For certain embodiments, the present invention involvesthe engineering of improved, Shiga toxin A Subunit effector polypeptidescomprising the combination of two or more of the following Shiga toxineffector polypeptide sub-regions: (1) a de-immunized sub-region, (2) aprotease-cleavage resistant sub-region near the carboxy-terminus of aShiga toxin A1 fragment region, and (3) a T-cell epitope-peptideembedded or inserted sub-region. For example, the Shiga toxin effectorpolypeptide of the present invention may comprise the combination of:(1) a de-immunized sub-region, (2) a protease-cleavage resistantsub-region near the carboxy-terminus of a Shiga toxin A1 fragmentregion, and (3) a T-cell epitope-peptide embedded or inserted sub-regionthat does not overlap with the de-immunized sub-region.

In certain embodiments, the cell-targeting molecule of the inventioncomprises a Shiga toxin effector polypeptide that comprises a Shigatoxin A1 fragment region, wherein the Shiga toxin A subunit effectorpolypeptide comprises: (a) an embedded or inserted, heterologous, CD8+T-cell epitope which disrupts an endogenous, B-cell and/or CD4+ T-cellepitope region (such as a region within the Shiga toxin A1 fragmentregion); (b) a disruption of at least three, endogenous, B-cell and/orCD4+ T-cell epitope regions (such as a three or more regions within theShiga toxin A1 fragment region) which do not overlap with the embeddedor inserted, heterologous, CD8+ T-cell epitope; and (c) a disruptedfurin-cleavage motif at the carboxy-terminus of the Shiga toxin A1fragment region; wherein the Shiga toxin A subunit effector polypeptideis capable of exhibiting a Shiga toxin effector function. In certainfurther embodiments, the Shiga toxin A subunit effector polypeptide istruncated at its carboxy-terminus, relative to a wild-type Shiga toxin Asubunit, resulting in the elimination of one or more endogenous, B-celland/or CD4+ T-cell epitope regions. In certain further embodiments, thefurin-cleavage motif comprises a carboxy-terminal truncation as comparedto the carboxy-terminus of a wild-type Shiga toxin A Subunit. In certainfurther embodiments, the furin-cleavage motif is disrupted by acarboxy-terminal truncation as compared to the carboxy-terminus of awild-type Shiga toxin A Subunit. For certain embodiments, thecell-targeting molecule is capable of exhibiting less relativeantigenicity and/or relative immunogenicity as compared to a referencemolecule, such as, e.g., a wild-type Shiga toxin A effector polypeptidecomprising a Shiga toxin A1 fragment region, a reference cell-targetingmolecule comprising a wild-type Shiga toxin A effector polypeptidecomprising a Shiga toxin A1 fragment region, or a referencecell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks any combination of the following features present in thecell targeting molecule: (1) an embedded or inserted, CD8+ T-cellepitope, (2) a disruption of at least three, endogenous, B-cell and/orCD4+ T-cell epitope regions, and/or (3) a disrupted furin-cleavage motifat the carboxy-terminus of the Shiga toxin A1 fragment region.

For purposes of the present invention, a Shiga toxin effectorpolypeptide is a polypeptide derived from a Shiga toxin A Subunit memberof the Shiga toxin family that is capable of exhibiting one or moreShiga toxin functions (see e.g., Cheung M et al., Mol Cancer 9: 28(2010); WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452;WO 2015/191764) and comprises a Shiga toxin A1 fragment derived regionhaving a carboxy-terminus. Shiga toxin functions include, e.g.,increasing cellular internalization, directing subcellular routing froman endosomal compartment to the cytosol, avoiding intracellulardegradation, catalytically inactivating ribosomes, and effectuatingcytostatic and/or cytotoxic effects.

The Shiga toxin family of protein toxins is composed of variousnaturally occurring toxins which are structurally and functionallyrelated, e.g., Shiga toxin, Shiga-like toxin 1, and Shiga-like toxin 2(Johannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). Holotoxinmembers of the Shiga toxin family contain targeting domains thatpreferentially bind a specific glycosphingolipid present on the surfaceof some host cells and an enzymatic domain capable of permanentlyinactivating ribosomes once inside a cell (Johannes L, Römer W, Nat RevMicrobiol 8: 105-16 (2010)). Members of the Shiga toxin family share thesame overall structure and mechanism of action (Engedal N et al.,Microbial Biotech 4: 32-46 (2011)). For example, Stx, SLT-1 and SLT-2display indistinguishable enzymatic activity in cell free systems (HeadS et al., J Biol Chem 266: 3617-21 (1991); Tesh V et al., Infect Immun61: 3392-402 (1993); Brigotti M et al., Toxicon 35:1431-1437 (1997)).

The Shiga toxin family encompasses true Shiga toxin (Stx) isolated fromS. dysenteriae serotype 1, Shiga-like toxin 1 A Subunit variants (SLT1or Stxl or SLT-1 or Slt-I) isolated from serotypes of enterohemorrhagicE. coli, and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2)isolated from serotypes of enterohemorrhagic E. coli. SLT1 differs byonly one amino acid residue from Stx, and both have been referred to asVerocytotoxins or Verotoxins (VTs) (O′Brien A, Curr Top MicrobiolImmunol 180: 65-94 (1992)). Although SLT1 and SLT2 variants are onlyabout 53-60% similar to each other at the primary amino acid sequencelevel, they share mechanisms of enzymatic activity and cytotoxicitycommon to the members of the Shiga toxin family (Johannes L, Römer W,Nat Rev Microbiol 8: 105-16 (2010)). Over 39 different Shiga toxins havebeen described, such as the defined subtypes Stx1a, Stx1c, Stx1d, andStx2a-g (Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)). Membersof the Shiga toxin family are not naturally restricted to any bacterialspecies because Shiga-toxin-encoding genes can spread among bacterialspecies via horizontal gene transfer (Strauch E et al., Infect Immun 69:7588-95 (2001); Bielaszewska M et al., Appl Environ Microbiol 73:3144-50 (2007); Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6(2011)). As an example of interspecies transfer, a Shiga toxin wasdiscovered in a strain of A. haemolyticus isolated from a patient(Grotiuz G et al., J Clin Microbiol 44: 3838-41 (2006)). Once a Shigatoxin encoding polynucleotide enters a new subspecies or species, theShiga toxin amino acid sequence is presumed to be capable of developingslight sequence variations due to genetic drift and/or selectivepressure while still maintaining a mechanism of cytotoxicity common tomembers of the Shiga toxin family (see Scheutz F et al., J ClinMicrobiol 50: 2951-63 (2012)).

1. De-Immunized, Shiga Toxin A Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention is de-immunized, such as, e.g., as compared to awild-type Shiga toxin, wild-type Shiga toxin polypeptide, and/or Shigatoxin effector polypeptide comprising only wild-type polypeptidesequences. The de-immunized, Shiga toxin effector polypeptides of thepresent invention each comprise a disruption of at least one (such as,e.g., at least two, three, four, five, six, seven, eight, nine or more),putative, endogenous, epitope region in order to reduce the antigenicand/or immunogenic potential of the Shiga toxin effector polypeptideafter administration of the polypeptide to a chordate. A Shiga toxineffector polypeptide and/or Shiga toxin A Subunit polypeptide, whethernaturally occurring or not, can be de-immunized by a method describedherein, described in WO 2015/113005 and/or WO 2015/113007, and/or knownto the skilled worker, wherein the resulting molecule retains orexhibits one or more Shiga toxin A Subunit functions.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises a disruption of an endogenous epitope orepitope region, such as, e.g., a B-cell and/or CD4+ T-cell epitope. Incertain embodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption of at least one (such as at least two,three, four, five, six, seven, eight or more) endogenous, B-cell and/orCD4+ T-cell epitope region. In certain embodiments, the Shiga toxineffector polypeptide of the present invention comprises a disruption ofat least one (such as at least two, three, four, five, six, seven, eightor more), endogenous, epitope region described herein, wherein thedisruption reduces the antigenic and/or immunogenic potential of theShiga toxin effector polypeptide after administration of the polypeptideto a chordate, and wherein the Shiga toxin effector polypeptide iscapable of exhibiting one or more Shiga toxin A Subunit functions, suchas, e.g., a significant level of Shiga toxin cytotoxicity. For example,the Shiga toxin effector polypeptide of the present invention comprisesa disruption of at least three, endogenous, B-cell and/or CD4+ T-cellepitope regions (such as, e.g., due to two or more mutations and one ormore truncations relative to a wild-type Shiga toxin A Subunit).

The term “disrupted” or “disruption” as used herein with regard to anepitope region refers to the deletion of at least one (such as at leasttwo, three, four, five, six, seven, eight or more) amino acid residue inan epitope region, inversion of two or more amino acid residues where atleast one of the inverted amino acid residues is in an epitope region,insertion of at least one (such as at least two, three, four, five, six,seven, eight or more) amino acid into an epitope region, and asubstitution of at least one amino acid residue in an epitope region. Anepitope region disruption by mutation includes amino acid substitutionswith non-standard amino acids and/or non-natural amino acids. Epitoperegions may alternatively be disrupted by mutations comprising themodification of an amino acid by the addition of a covalently-linkedchemical structure which masks at least one amino acid in an epitoperegion, see, e.g. PEGylation (see Zhang C et al., BioDrugs 26: 209-15(2012), small molecule adjuvants (Flower D, Expert Opin Drug Discov 7:807-17 (2012), and site-specific albumination (Lim S et al., J ControlRelease 207-93 (2015)).

Certain epitope regions and disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits provided in the Sequence Listing, noting that naturallyoccurring Shiga toxin A Subunits may comprise precursor forms containingsignal sequences of about 22 amino acids at their amino-terminals whichare removed to produce mature Shiga toxin A Subunits and arerecognizable to the skilled worker. Further, certain epitope regiondisruptions are indicated herein by reference to specific amino acids(e.g. S for a serine residue) natively present at specific positionswithin native Shiga toxin A Subunits (e.g. S33 for the serine residue atposition 33 from the amino-terminus) followed by the amino acid withwhich that residue has been substituted in the particular mutation underdiscussion (e.g. S33I represents the amino acid substitution ofisoleucine for serine at amino acid residue 33 from the amino-terminus).

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises a disruption of at leastone (such as at least two, three, four, five, six, seven, eight or more)epitope region provided herein. For example, the de-immunized, Shigatoxin effector polypeptide of the present invention may comprise adisruption of at least three epitope regions provided herein. In certainembodiments, the de-immunized, Shiga toxin effector polypeptide of thepresent invention comprises a disruption of at least four epitoperegions provided herein. In certain embodiments, the de-immunized, Shigatoxin effector polypeptide of the present invention comprises adisruption of at least five epitope regions provided herein. In certainembodiments, the de-immunized, Shiga toxin effector polypeptide of thepresent invention comprises a disruption of at least one epitope regiondescribed in WO 2015/113005 or WO 2015/113007. As described herein, whenthe Shiga toxin effector polypeptide also comprises an embedded orinserted, heterologous, CD8+ T-cell epitope, at least some number ofdisrupted, endogenous, B-cell and/or CD4+ T-cell epitope region does notoverlap with the embedded or inserted, heterologous, CD8+ T-cellepitope.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises, consists of, or consistsessentially of a full-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ IDNO:1), StxA (SEQ ID NO:2), or SLT-2A (SEQ ID NO:3)) comprising at leastone disruption of the amino acid sequence selected from the group ofnatively positioned amino acids consisting of: 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 210-218 of SEQ ID NO:3; 240-258 of SEQ ID NO:3; 243-257 of SEQ IDNO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 ofSEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQID NO:2, or the equivalent position in a Shiga toxin A Subunitpolypeptide, conserved Shiga toxin effector polypeptide sub-region,and/or non-native, Shiga toxin effector polypeptide sequence (such asthe Shiga toxin effector polypeptides shown in SEQ ID NOs: 4-18).

In certain embodiments, the de-immunized Shiga toxin effectorpolypeptide of the present invention comprises, consists essentially of,or consists of a full-length or truncated Shiga toxin A Subunit (e.g.SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), SLT-2A (SEQ ID NO:3), or anyone of SEQ ID NOs: 7-18 further comprising a disruption of at least one(such as at least two, three, four, five, six, seven, eight or more)endogenous, B-cell and/or CD4+ T-cell epitope region, wherein the B-cellregion is selected from the group of natively positioned Shiga toxin ASubunit regions consisting of: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ IDNO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ IDNO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ IDNO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; orthe equivalent region in a Shiga toxin A Subunit or derivative thereof(such as the equivalent region in any one of the Shiga toxin 1 A Subunitvariants shown in SEQ ID NOs: 4-6 and the Shiga-like toxin 2 A Subunitvariants shown in SEQ ID NOs: 7-18); and the CD4+ T-cell epitope regionis selected from the group of natively positioned Shiga toxin A Subunitregions consisting of: 4-33 of SEQ ID NO:1 or SEQ ID NO:2; 34-78 of SEQID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 or SEQ ID NO:2; 128-168 ofSEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ ID NO:1 or SEQ ID NO:2;236-258 of SEQ ID NO:1 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQID NO:2; or the equivalent region in a Shiga toxin A Subunit orderivative thereof (such as the equivalent region in any one of theShiga toxin 1 A Subunit variants shown in SEQ ID NOs: 4-6 and theShiga-like toxin 2 A Subunit variants shown in SEQ ID NOs: 7-18). Incertain embodiments, the B-cell epitope region is selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3;179-190 of SEQ ID NO: 1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 ofSEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 210-218 of SEQ ID NO:3and 243-257 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalent region ina Shiga toxin A Subunit or derivative thereof (such as the equivalentregion in any one of the Shiga toxin 1 A Subunit variants shown in SEQID NOs: 4-6 and the Shiga-like toxin 2 A Subunit variants shown in SEQID NOs: 7-18); and the CD4+ T-cell epitope region is selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 4-33 of SEQ ID NO:1 or SEQ ID NO:2; 34-78 of SEQ ID NO: 1 or SEQ IDNO:2; 77-103 of SEQ ID NO: 1 or SEQ ID NO:2; 128-168 of SEQ ID NO: 1 orSEQ ID NO:2; 160-183 of SEQ ID NO: 1 or SEQ ID NO:2; and 236-258 of SEQID NO: 1 or SEQ ID NO:2;or the equivalent region in a Shiga toxin ASubunit or derivative thereof (such as the equivalent region in any oneof the Shiga toxin 1 A Subunit variants shown in SEQ ID NOs: 4-6 and theShiga-like toxin 2 A Subunit variants shown in SEQ ID NOs: 7-18).

In certain embodiments, the de-immunized Shiga toxin effectorpolypeptide of the present invention comprises, consists essentially of,or consists of a full-length or truncated Shiga toxin A Subunit (e.g.SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), Shiga toxin 1 A Subunitvariant effector polypeptide (SEQ ID NOs: 4-6), SLT-2A (SEQ ID NO:3), orShiga-like toxin 2 A Subunit variant effector polypeptide (SEQ ID NOs:7-18)) comprising a disruption of at least three, endogenous, B-celland/or CD4+ T-cell epitope regions, wherein the disruption comprises amutation, relative to a wild-type Shiga toxin A Subunit, in the B-cellepitope region selected from the group of natively positioned Shigatoxin A Subunit regions consisting of: 1-15 of SEQ ID NO:1 or SEQ IDNO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ IDNO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ IDNO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 210-218 of SEQ ID NO:3 and 243-257 of SEQ ID NO: 1 or SEQ ID NO:2;or the equivalent region in a Shiga toxin A Subunit or derivativethereof (such as the equivalent region in any one of the Shiga toxin 1 ASubunit variants shown in SEQ ID NOs: 4-6 and Shiga-like toxin 2 ASubunit variants shown in SEQ ID NOs: 7-18); and/or the CD4+ T-cellepitope region selected from the group of natively positioned Shigatoxin A Subunit regions consisting of: 4-33 of SEQ ID NO: 1 or SEQ IDNO:2; 34-78 of SEQ ID NO: 1 or SEQ ID NO:2; 77-103 of SEQ ID NO: 1 orSEQ ID NO:2; 128-168 of SEQ ID NO: 1 or SEQ ID NO:2; 160-183 of SEQ IDNO: 1 or SEQ ID NO:2; and 236-258 of SEQ ID NO: 1 or SEQ ID NO:2;or theequivalent region in a Shiga toxin A Subunit or derivative thereof (suchas the equivalent region in any one of the Shiga toxin 1 A Subunitvariants shown in SEQ ID NOs: 4-6 and the Shiga-like toxin 2 A Subunitvariants shown in SEQ ID NOs: 7-18). In certain embodiments, each of theat least three of the B-cell and/or CD4+ T-cell epitope regionscomprises a disruption comprising an amino acid residue substitutionrelative to a wild-type Shiga toxin A Subunit sequence.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises, consists of, or consists essentially of atruncated Shiga toxin A Subunit. Truncations of Shiga toxin A Subunitsmight result in the deletion of an entire epitope region(s) withoutaffecting Shiga toxin effector function(s). The smallest, Shiga toxin ASubunit fragment shown to exhibit significant enzymatic activity was apolypeptide composed of residues 75-247 of StxA (Al-Jaufy A et al.,Infect Immun 62: 956-60 (1994)). Truncating the carboxy-terminus ofSLT-1A, StxA, or SLT-2A to amino acids 1-251 removes two predictedB-cell epitope regions, two predicted CD4 positive (CD4+) T-cellepitopes, and a predicted, discontinuous, B-cell epitope. Truncating theamino-terminus of SLT-1A, StxA, or SLT-2A to 75-293 removes at leastthree, predicted, B-cell epitope regions and three predicted CD4+ T-cellepitopes. Truncating both amino- and carboxy-terminals of SLT-1A, StxA,or SLT-2A to 75-251 deletes at least five, predicted, B-cell epitoperegions; four, putative, CD4+ T-cell epitopes; and one, predicted,discontinuous, B-cell epitope.

In certain embodiments, a Shiga toxin effector polypeptide of theinvention may comprise, consist of, or consist essentially of afull-length or truncated Shiga toxin A Subunit with at least one (suchas at least two, three, four, five, six, seven, eight or more) mutation,e.g. deletion, insertion, inversion, or substitution, in a providedepitope region. In certain further embodiments, the polypeptidescomprise a disruption which comprises a deletion of at least one aminoacid within the epitope region. In certain further embodiments, thepolypeptides comprise a disruption which comprises an insertion of atleast one amino acid within the epitope region. In certain furtherembodiments, the polypeptides comprise a disruption which comprises aninversion of amino acids, wherein at least one inverted amino acid iswithin the epitope region. In certain further embodiments, thepolypeptides comprise a disruption which comprises a substitution of atleast one (such as at least two, three, four, five, six, seven, eight ormore) amino acid within the epitope region. In certain furtherembodiments, the polypeptides comprise a disruption which comprises amutation, such as an amino acid substitution to a non-standard aminoacid or an amino acid with a chemically modified side chain. Numerousexamples of single amino acid substitutions are provided in the Examplesbelow.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention may comprise, consist of, or consist essentially of afull-length or truncated Shiga toxin A Subunit with one or moremutations as compared to the native sequence which comprises at leastone amino acid substitution selected from the group consisting of: A, G,V, L, I, P, C, M, F, S, D, N, Q, H, and K. In certain furtherembodiments, the polypeptide may comprise, consist of, or consistessentially of a full-length or truncated Shiga toxin A Subunit with asingle mutation as compared to the native sequence wherein thesubstitution is selected from the group consisting of: D to A, D to G, Dto V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, Eto L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, Gto A, H to A, H to G, H to V, H to L, H to I, H to F, H to M, K to A, Kto G, K to V, K to L, K to I, K to M, K to H, L to A, L to G, N to A, Nto G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, Rto G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, Rto H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, Tto G, T to V, T to L, T to I, T to F, T to M, T to S, Y to A, Y to G, Yto V, Y to L, Y to I, Y to F, and Y to M.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention comprise, consist of, or consist essentially of a full-lengthor truncated Shiga toxin A Subunit with one or more mutations ascompared to the native amino acid residue sequence which comprises atleast one amino acid substitution of an immunogenic residue and/orwithin an epitope region, wherein at least one substitution occurs atthe natively positioned group of amino acids selected from the groupconsisting of: 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ IDNO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1or SEQ ID NO:2; 46 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 orSEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ IDNO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1 orSEQ ID NO:2; 57 of SEQ ID NO:1 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 ofSEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQID NO:1 or SEQ ID NO:2; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;96 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 orSEQ ID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1 orSEQ ID NO:2; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141 of SEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 orSEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ IDNO:1 or SEQ ID NO:2; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ IDNO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ IDNO:2, or the equivalent position in a Shiga toxin A Subunit polypeptide,conserved Shiga toxin effector polypeptide sub-region, and/ornon-native, Shiga toxin effector polypeptide sequence (such as the Shigatoxin 1 A Subunit variant effector polypeptides shown in SEQ ID NOs: 4-6or the Shiga-like toxin 2 A Subunit variant effector polypeptides shownin SEQ ID NOs: 7-18).

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise, consist of, or consist essentially of afull-length or truncated Shiga toxin A Subunit with at least onesubstitution of an immunogenic residue and/or within an epitope region,wherein at least one amino acid substitution is to a non-conservativeamino acid (see, e.g., Table C, infra) relative to a natively occurringamino acid positioned at one of the following native positions: 1 of SEQID NO:1 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 ofSEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 ofSEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ IDNO:1 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ IDNO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ IDNO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1 or SEQ IDNO:2; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 ofSEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1 or SEQ ID NO:2; 108 ofSEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1 or SEQ IDNO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ IDNO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 orSEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2;186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 orSEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 orSEQ ID NO:2; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 orSEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 orSEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2, or theequivalent position in a Shiga toxin A Subunit polypeptide, conservedShiga toxin effector polypeptide sub-region, and/or non-native, Shigatoxin effector polypeptide sequence (such as the Shiga toxin effectorpolypeptide of any one of SEQ ID NOs: 4-18).

In certain embodiments, the Shiga toxin effector polypeptides of theinvention comprise, consist essentially of, or consist of a full-lengthor truncated Shiga toxin A Subunit with one or more mutations ascompared to the native amino acid residue sequence which comprises atleast one amino acid substitution of an immunogenic residue and/orwithin an epitope region, wherein at least one substitution occurs atthe natively positioned amino acid position selected from the groupconsisting of: 1 of SEQ ID NO:1 or SEQ ID NO:2; 11 of SEQ ID NO:1 or SEQID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1, SEQ IDNO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2;59 of SEQ ID NO:1, SEQ ID NO:2; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 ofSEQ ID NO:1 or SEQ ID NO:2; 110 of SEQ ID NO:1 or SEQ ID NO:2; 141 ofSEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1 or SEQ ID NO:2; 188 ofSEQ ID NO:1 or SEQ ID NO:2; 242 of SEQ ID NO:1 or SEQ ID NO:2; 248 ofSEQ ID NO:1 or SEQ ID NO:2; and 251 of SEQ ID NO:1 or SEQ ID NO:2.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one amino acidsubstitution selected from the group consisting of: K1 to A, G, V, L, I,F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S,and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, and S;T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 toA, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, and S; S33 to A,G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to Aand P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;I57 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F,S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 toA, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H;V88 to A and G; I88 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F,M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 toA, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A;D111 to A, G, V, L, I, F, S, and Q; S112 to A, G, V, L, I, F, and M;D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G; R179 toA, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S;T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q;D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G,V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, andH; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S, and Q;D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S,K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V,and S; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M;R247 to A, G, V, L, I, F, M, Q, S, K, and H; R248 to A, G, V, L, I, F,M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 toA, G, V, L, I, F, M, Q, S, K, and H; C262 to A, G, V, and S; D264 to A,G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, andS.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise, consist of, or consist essentially of afull-length or truncated Shiga toxin A Subunit with at least one (suchas at least two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen or more) of the following amino acidsubstitutions: K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K,S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V,N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L,G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A,E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M,S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G,T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F,G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I,Y247A, R247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/or T286I.In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise, consist essentially of, or consist of afull-length or truncated Shiga toxin A Subunit with at least one (suchas at least two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen or more) of the following amino acidsubstitutions: K1A, S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A,D141A, G147A, R188A, C242S, R248A, and R251A. These epitope disruptingsubstitutions may be combined to form a de-immunized, Shiga toxineffector polypeptide with multiple substitutions per epitope regionand/or multiple epitope regions disrupted while still retaining Shigatoxin effector function. For example, substitutions at the nativelypositioned K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K,S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V,N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L,G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A,E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M,S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A, T180G,T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F,G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I,Y247A, R247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/or T286Imay be combined, where possible, with substitutions at the nativelypositioned residues K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H,T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G,N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V,R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R,E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P,L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A,T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A,S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S,S247I, Y247A, R247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/orT286I to create de-immunized, Shiga toxin effector polypeptides of theinvention. For example, the Shiga toxin effector polypeptides of theinvention may comprise, consist essentially of, or consist of afull-length or truncated Shiga toxin A Subunit comprising the followingsubstitutions at native positions in a Shiga toxin A Subunit: K1A, S45I,V54I, R55L, I57F, P59F, E60T, E61L, G110A, G147A, C242S, R248A, andR251A. These substitutions correspond to those present in the Shigatoxin effector polypeptide of the exemplary cell-targeting moleculeshown in any one of SEQ ID NOs: 24-27 and 97-100. For example, the Shigatoxin effector polypeptides of the invention may comprise, consistessentially of, or consist of a full-length or truncated Shiga toxin ASubunit comprising the following substitutions at native positions in aShiga toxin A Subunit: S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A,R188A, C242S, R248A, and R251A. These substitutions correspond to thosepresent in the Shiga toxin effector polypeptide of the exemplarycell-targeting molecule shown in any one of SEQ ID NOs: 28-29, 31-32,34, 36, 101-102, 104-105, 106, and 108. For example, the Shiga toxineffector polypeptides of the invention may comprise, consist essentiallyof, or consist of a full-length or truncated Shiga toxin A Subunitcomprising the following substitutions at native positions in a Shigatoxin A Subunit: S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A, D141A,R188A, C242S, R248A, and R251A. These substitutions correspond to thosepresent in the Shiga toxin effector polypeptide of the exemplarycell-targeting molecule shown in any one of SEQ ID NOs: 30 or 103.

Any of the de-immunized, Shiga toxin effector polypeptide sub-regionsand/or epitope disrupting mutations described herein may be used aloneor in combination with each individual embodiment of the presentinvention, including methods of the present invention.

2. Protease-Cleavage Resistant, Shiga Toxin A Subunit EffectorPolypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derived regionhaving a carboxy-terminus and (2) a disrupted furin-cleavage motif atthe carboxy-terminus of the Shiga toxin A1 fragment region. Improvingthe stability of connections between the Shiga toxin component and othercomponents of cell-targeting molecules, e.g., cell-targeting bindingregions, can improve their toxicity profiles after administration toorganisms by reducing non-specific toxicities caused by the breakdown ofthe connection and loss of cell-targeting, such as, e.g., as a result ofproteolysis. In certain embodiments, the protease-cleavage resistantShiga toxin effector polypeptide of the present invention has acarboxy-terminal truncation as compared to the carboxy-terminus of awild-type Shiga toxin A Subunit.

Shiga toxin A Subunits of members of the Shiga toxin family comprise aconserved, furin-cleavage site at the carboxy-terminal of their A1fragment regions important for Shiga toxin function. Furin-cleavage sitemotifs and furin-cleavage sites can be identified by the skilled workerusing standard techniques and/or by using the information herein.

The model of Shiga toxin cytotoxicity is that intracellular proteolyticprocessing of Shiga toxin A Subunits by furin in intoxicated cells isessential for 1) liberation of the A1 fragment from the rest of theShiga holotoxin, 2) escape of the A1 fragment from the endoplasmicreticulum by exposing a hydrophobic domain in the carboxy-terminus ofthe A1 fragment, and 3) enzymatic activation of the A1 fragment (seeJohannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). The efficientliberation of the Shiga toxin A1 fragment from the A2 fragment and therest of the components of the Shiga holotoxin in the endoplasmicreticulum of intoxicated cells is essential for efficient intracellularrouting to the cytosol, maximal enzymatic activity, efficient ribosomeinactivation, and achieving optimal cytotoxicity, i.e. comparable to awild-type Shiga toxin (see e.g. WO 2015/191764 and references therein).

During Shiga toxin intoxication, the A Subunit is proteolyticallycleaved by furin at the carboxy bond of a conserved arginine residue(e.g. the arginine residue at position 251 in StxA and SLT-1A and thearginine residue at position 250 in Stx2A and SLT-2A). Furin cleavage ofShiga toxin A Subunits occurs in endosomal and/or Golgi compartments.Furin is a specialized serine endoprotease which is expressed by a widevariety of cell types, in all human tissues examined, and by most animalcells. Furin cleaves polypeptides comprising accessible motifs oftencentered on the minimal, dibasic, consensus motif R-x-(R/K/x)-R. The ASubunits of members of the Shiga toxin family comprise a conserved,surface-exposed, extended loop structure (e.g. 242-261 in StxA andSLT-1A, and 241-260 in SLT-2) with a conserved S-R/Y-x-x-R motif whichis cleaved by furin. The surface exposed, extended loop structurepositioned at amino acid residues 242-261 in StxA is required forfurin-induced cleavage of StxA, including features flanking the minimal,furin-cleavage motif R-x-x-R.

Furin-cleavage motifs and furin-cleavage sites in Shiga toxin A Subunitsand Shiga toxin effector polypeptides can be identified by the skilledworker using standard methods and/or by using the information herein.Furin cleaves the minimal, consensus motif R-x-x-R (Schalken J et al., JClin Invest 80: 1545-9 (1987); Bresnahan P et al., J CellBiol 111:2851-9 (1990); Hatsuzawa K et al., J Biol Chem 265: 22075-8 (1990); WiseR et al., Proc Natl Acad Sci USA 87: 9378-82 (1990); Molloy S et al., JBiol Chem 267: 16396-402 (1992)). Consistent with this, many furininhibitors comprise peptides comprising the motif R-x-x-R. An example ofa synthetic inhibitor of furin is a molecule comprising the peptideR-V-K-R (SEQ ID NO:157) (Henrich S et al., Nat Struct Biol 10: 520-6(2003)). In general, a peptide or protein comprising a surfaceaccessible, dibasic amino acid motif with two positively charged, aminoacids separated by two amino acid residues may be predicted to besensitive to furin-cleavage with cleavage occurring at the carboxy bondof the last basic amino acid in the motif.

Consensus motifs in substrates cleaved by furin have been identifiedwith some degree of specificity. A furin-cleavage site motif has beendescribed that comprises a region of twenty, continuous, amino acidresidues, which can be labeled P14 through P6′ (Tian S et al., Int J MolSci 12: 1060-5 (2011)) using the nomenclature described in Schechter I,Berger, A, Biochem Biophys Res Commun 32: 898-902 (1968). According tothis nomenclature, the furin-cleavage site is at the carboxy bond of theamino acid residue designated P1, and the amino acid residues of thefurin-cleavage motif are numbered P2, P3, P4, etc., in the directiongoing toward the amino-terminus from this reference P1 residue. Theamino acid residues of the motif going toward the carboxy-terminus fromthe P1 reference residue are numbered with the prime notation P2′, P3′,P4′, etc. Using this nomenclature, the P6 to P2′ region delineates thecore substrate of the furin cleavage motif which is bound by theenzymatic domain of furin. The two flanking regions P14 to P7 and P3′ toP6′ are often rich in polar, amino acid residues to increase theaccessibility to the core furin cleavage site located between them.

A general, furin-cleavage site is often described by the consensus motifR-x-x-R which corresponds to P4-P3-P2-P1; where “R” represents anarginine residue (see Table A, supra), a dash “-” represents a peptidebond, and a lowercase “x” represents any amino acid residue. However,other residues and positions may help to further define furin-cleavagemotifs. A slightly more refined furin-cleavage site, consensus motif isoften reported as the consensus motif R-x-[K/R]-R (where a forward slash“/” means “or” and divides alternative amino acid residues at the sameposition), which corresponds to P4-P3-P2-P1, because it was observedthat furin has a strong preference for cleaving substrates containingthis motif.

In addition to the minimal, furin-cleavage site R-x-x-R, a larger,furin-cleavage motif has been described with certain amino acid residuepreferences at certain positions. By comparing various known furinsubstrates, certain physicochemical properties have been characterizedfor the amino acid residues in a 20 amino acid residue long,furin-cleavage site motif. The P6 to P2′ region of the furin-cleavagemotif delineates the core furin-cleavage site which physically interactswith the enzymatic domain of furin. The two flanking regions P14 to P7and P3′ to P6′ are often hydrophilic being rich in polar, amino acidresidues to increase the surface accessibility of the corefurin-cleavage site located between them.

In general, the furin-cleavage motif region from position P5 to P1 tendsto comprise amino acid residues with a positive charge and/or highisoelectric points. In particular, the P1 position, which marks theposition of furin proteolysis, is generally occupied by an arginine butother positively charged, amino acid residues may occur in thisposition. Positions P2 and P3 tend to be occupied by flexible, aminoacid residues, and in particular P2 tends to be occupied by arginine,lysine, or sometimes by very small and flexible amino acid residues likeglycine. The P4 position tends to be occupied by positively charged,amino acid residues in furin substrates. However, if the P4 position isoccupied by an aliphatic, amino acid residue, then the lack of apositively charged, functional group can be compensated for by apositively charged residue located at position(s) P5 and/or P6.Positions P1′ and P2′ are commonly occupied by aliphatic and/orhydrophobic amino acid residues, with the P1′ position most commonlybeing occupied by a serine.

The two, hydrophilic, flanking regions tend to be occupied by amino acidresidues which are polar, hydrophilic, and have smaller amino acidfunctional groups; however, in certain verified furin substrates, theflanking regions do not contain any hydrophilic, amino acid residues(see Tian S, Biochem Insights 2: 9-20 (2009)).

The twenty amino acid residue, furin-cleavage motif and furin-cleavagesite found in native, Shiga toxin A Subunits at the junction between theShiga toxin A1 fragment and A2 fragment is well characterized in certainShiga toxins. For example in StxA (SEQ ID NO:2) and SLT-1A (SEQ IDNO: 1) or another Shiga toxin 1 A Subunit effector polypeptide (e.g. SEQID NOs: 4-6), this furin-cleavage motif is natively positioned from L238to F257, and in SLT-2A (SEQ ID NO:3 or Shiga toxin effector polypeptidesbased on Shiga-like toxin 2 A Subunit variants (e.g. SEQ ID NOs: 7-18),this furin-cleavage motif is natively positioned from V237 to Q256.Based on amino acid homology, experiment, and/or furin-cleavage assaysdescribed herein, the skilled worker can identify furin-cleavage motifsin other native, Shiga toxin A Subunits or Shiga toxin effectorpolypeptides, where the motifs are actual furin-cleavage motifs or arepredicted to result in the production of A1 and A2 fragments after furincleavage of those molecules within a eukaryotic cell.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment derivedpolypeptide. The carboxy-terminus of a Shiga toxin A1 fragment derivedpolypeptide may be identified by the skilled worker by using techniquesknown in the art, such as, e.g., by using protein sequence alignmentsoftware to identify (i) a furin-cleavage motif conserved with anaturally occurring Shiga toxin, (ii) a surface exposed, extended loopconserved with a naturally occurring Shiga toxin, and/or (iii) a stretchof amino acid residues which are predominantly hydrophobic (i.e. ahydrophobic “patch”) that may be recognized by the ERAD system.

A protease-cleavage resistant, Shiga toxin effector polypeptide of thepresent invention (1) may be completely lacking any furin-cleavage motifat a carboxy-terminus of its Shiga toxin A1 fragment region and/or (2)comprise a disrupted furin-cleavage motif at the carboxy-terminus of itsShiga toxin A1 fragment region and/or region derived from thecarboxy-terminus of a Shiga toxin A1 fragment. A disruption of afurin-cleavage motif include various alterations to an amino acidresidue in the furin-cleavage motif, such as, e.g., a post-translationmodification(s), an alteration of one or more atoms in an amino acidfunctional group, the addition of one or more atoms to an amino acidfunctional group, the association to a non-proteinaceous moiety(ies),and/or the linkage to an amino acid residue, peptide, polypeptide suchas resulting in a branched proteinaceous structure.

Protease-cleavage resistant, Shiga toxin effector polypeptides may becreated from a Shiga toxin effector polypeptide and/or Shiga toxin ASubunit polypeptide, whether naturally occurring or not, using a methoddescribed herein, described in WO 2015/191764, and/or known to theskilled worker, wherein the resulting molecule still retains one or moreShiga toxin A Subunit functions.

For purposes of the present invention with regard to a furin-cleavagesite or furin-cleavage motif, the term “disruption” or “disrupted”refers to an alteration from the naturally occurring furin-cleavage siteand/or furin-cleavage motif, such as, e.g., a mutation, that results ina reduction in furin-cleavage proximal to the carboxy-terminus of aShiga toxin A1 fragment region, or identifiable region derived thereof,as compared to the furin-cleavage of a wild-type Shiga toxin A Subunitor a polypeptide derived from a wild-type Shiga toxin A Subunitcomprising only wild-type polypeptide sequences. An alteration to anamino acid residue in the furin-cleavage motif includes a mutation inthe furin-cleavage motif, such as, e.g., a deletion, insertion,inversion, substitution, and/or carboxy-terminal truncation of thefurin-cleavage motif, as well as a post-translation modification, suchas, e.g., as a result of glycosylation, albumination, and the like whichinvolve conjugating or linking a molecule to the functional group of anamino acid residue. Because the furin-cleavage motif is comprised ofabout twenty, amino acid residues, in theory, alterations,modifications, mutations, deletions, insertions, and/or truncationsinvolving one or more amino acid residues of any one of these twentypositions might result in a reduction of furin-cleavage sensitivity(Tian S et al., Sci Rep 2: 261 (2012)). The disruption of afurin-cleavage site and/or furin-cleavage motif may or may not increaseresistance to cleavage by other proteases, such as, e.g., trypsin andextracellular proteases common in the vascular system of mammals. Theeffects of a given disruption to cleavage sensitivity of a givenprotease may be tested by the skilled worker using techniques known inthe art.

For purposes of the present invention, a “disrupted furin-cleavagemotif” is furin-cleavage motif comprising an alteration to one or moreamino acid residues derived from the 20 amino acid residue regionrepresenting a conserved, furin-cleavage motif found in native, Shigatoxin A Subunits at the junction between the Shiga toxin A1 fragment andA2 fragment regions and positioned such that furin cleavage of a Shigatoxin A Subunit results in the production of the A1 and A2 fragments;wherein the disrupted furin-cleavage motif exhibits reduced furincleavage in an experimentally reproducible way as compared to areference molecule comprising a wild-type, Shiga toxin A1 fragmentregion fused to a carboxy-terminal polypeptide of a size large enough tomonitor furin cleavage using the appropriate assay known to the skilledworker and/or described herein.

Examples of types of mutations which can disrupt a furin-cleavage siteand furin-cleavage motif are amino acid residue deletions, insertions,truncations, inversions, and/or substitutions, including substitutionswith non-standard amino acids and/or non-natural amino acids. Inaddition, furin-cleavage sites and furin-cleavage motifs can bedisrupted by mutations comprising the modification of an amino acid bythe addition of a covalently-linked structure which masks at least oneamino acid in the site or motif, such as, e.g., as a result ofPEGylation, the coupling of small molecule adjuvants, and/orsite-specific albumination.

If a furin-cleavage motif has been disrupted by mutation and/or thepresence of non-natural amino acid residues, certain disruptedfurin-cleavage motifs may not be easily recognizable as being related toany furin-cleavage motif; however, the carboxy-terminus of the Shigatoxin A1 fragment derived region will be recognizable and will definewhere the furin-cleavage motif would be located were it not disrupted.For example, a disrupted furin-cleavage motif may comprise less than thetwenty, amino acid residues of the furin-cleavage motif due to acarboxy-terminal truncation as compared to a Shiga toxin A Subunitand/or Shiga toxin A1 fragment.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment polypeptideregion; wherein the Shiga toxin effector polypeptide (and anycell-targeting molecule comprising it) is more furin-cleavage resistantas compared to a reference molecule, such as, e.g., a wild-type Shigatoxin polypeptide comprising the carboxy-terminus of an A1 fragmentand/or the conserved, furin-cleavage motif between A1 and A2 fragments.For example, a reduction in furin cleavage of one molecule compared to areference molecule may be determined using an in vitro, furin-cleavageassay described in the Examples below, conducted using the sameconditions, and then performing a quantitation of the band density ofany fragments resulting from cleavage to quantitatively measure inchange in furin cleavage.

In certain embodiments, the Shiga toxin effector polypeptide is moreresistant to furin-cleavage in vitro and/or in vivo as compared to awild-type, Shiga toxin A Subunit.

In general, the protease-cleavage sensitivity of a cell-targetingmolecule of the present invention is tested by comparing it to the samemolecule having its furin-cleavage resistant, Shiga toxin effectorpolypeptide replaced with a wild-type, Shiga toxin effector polypeptidecomprising a Shiga toxin A1 fragment. In certain embodiments, themolecules of the present invention comprising a disrupted furin-cleavagemotif exhibits a reduction in in vitro furin cleavage of 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98% or greater compared to a referencemolecule comprising a wild-type, Shiga toxin A1 fragment fused at itscarboxy-terminus to a peptide or polypeptide, such as, e.g., thereference molecule SLT-1A-WT::scFv-1 described in Example 2, below.

Several furin-cleavage motif disruptions have been described. Forexample, mutating the two conserved arginines to alanines in the minimalR-x-x-R motif completely blocked processing by furin and/or furin-likeproteases (see e.g Duda A et al., J Virology 78: 13865-70 (2004)).Because the furin-cleavage site motif is comprised of about twenty aminoacid residues, in theory, certain mutations involving one or more of anyone of these twenty, amino acid residue positions might abolish furincleavage or reduce furin cleavage efficiency (see e.g. Tian S et al.,Sci Rep 2: 261 (2012)).

In certain embodiments, the molecules of the present invention comprisea Shiga toxin effector polypeptide derived from at least one A Subunitof a member of the Shiga toxin family wherein the Shiga toxin effectorpolypeptide comprises a disruption in one or more amino acids derivedfrom the conserved, highly accessible, protease-cleavage sensitive loopof Shiga toxin A Subunits. For example, in StxA and SLT-1A, this highlyaccessible, protease-sensitive loop is natively positioned from aminoacid residues 242 to 261, and in SLT-2A, this conserved loop is nativelypositioned from amino acid residues 241 to 260. Based on polypeptidesequence homology, the skilled worker can identify this conserved,highly accessible loop structure in other Shiga toxin A Subunits.Certain mutations to the amino acid residues in this loop can reduce theaccessibility of certain amino acid residues within the loop toproteolytic cleavage and this might reduce furin-cleavage sensitivity.

In certain embodiments, a molecule of the present invention comprises aShiga toxin effector polypeptide comprising a disrupted furin-cleavagemotif comprising a mutation in the surface-exposed, protease sensitiveloop conserved among Shiga toxin A Subunits. In certain furtherembodiments, a molecule of the present invention comprises a Shiga toxineffector polypeptide comprising a disrupted furin-cleavage motifcomprising a mutation in this protease-sensitive loop of Shiga toxin ASubunits, the mutation which reduce the surface accessibility of certainamino acid residues within the loop such that furin-cleavage sensitivityis reduced.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in terms of existence, position, or functional group of oneor both of the consensus amino acid residues P1 and P4, such as, e.g.,the amino acid residues in positions 1 and 4 of the minimalfurin-cleavage motif R/Y-x-x-R. For example, mutating one or both of thetwo arginine residues in the minimal, furin consensus site R-x-x-R toalanine will disrupt a furin-cleavage motif and prevent furin-cleavageat that site. Similarly, amino acid residue substitutions of one or bothof the arginine residues in the minimal furin-cleavage motif R-x-x-R toany non-conservative amino acid residue known to the skilled worker willreduced the furin-cleavage sensitivity of the motif. In particular,amino acid residue substitutions of arginine to any non-basic amino acidresidue which lacks a positive charge, such as, e.g., A, G, P, S, T, D,E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disruptedfurin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in the spacing between the consensus amino acid residues P4and P1 in terms of the number of intervening amino acid residues beingother than two, and, thus, changing either P4 and/or P1 into a differentposition and eliminating the P4 and/or P1 designations. For example,deletions within the furin-cleavage motif of the minimal furin-cleavagesite or the core, furin-cleavage motif will reduce the furin-cleavagesensitivity of the furin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif comprises oneor more amino acid residue substitutions, as compared to a wild-type,Shiga toxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residuesubstitutions within the minimal furin-cleavage site R/Y-x-x-R, such as,e.g., for StxA and SLT-1A (and other Shiga toxin 1 A Subunit variants)derived Shiga toxin effector polypeptides, the natively positioned aminoacid residue R248 substituted with any non-positively charged, aminoacid residue and/or R251 substituted with any non-positively charged,amino acid residue; and for SLT-2A (and other Shiga-like toxin 2 ASubunit variants) derived Shiga toxin effector polypeptides, thenatively positioned amino acid residue R/Y247 substituted with anynon-positively charged, amino acid residue and/or R250 substituted withany non-positively charged, amino acid residue. In certain furtherembodiments, the disrupted furin-cleavage motif comprises one or moreamino acid residue substitutions within the minimal furin-cleavage siteR/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxineffector polypeptides (and other Shiga toxin 1 A Subunit variants), thenatively positioned amino acid residues R248 and R251 are substitutedwith an alanine residue; and for SLT-2A derived Shiga toxin effectorpolypeptides (and other Shiga-like toxin 2 A Subunit variants), thenatively positioned amino acid residues R/Y247 and R250 substituted withan alanine residue.

In certain embodiments, the disrupted furin-cleavage motif comprises anun-disrupted, minimal furin-cleavage site R/Y-x-x-R but insteadcomprises a disrupted flanking region, such as, e.g., amino acid residuesubstitutions in one or more amino acid residues in the furin-cleavagemotif flanking regions natively position at, e.g., 241-247 and/or252-259. In certain further embodiments, the disrupted furin cleavagemotif comprises a substitution of one or more of the amino acid residueslocated in the P1-P6 region of the furin-cleavage motif; mutating P1′ toa bulky amino acid, such as, e.g., R, W, Y, F, and H; and mutating P2′to a polar and hydrophilic amino acid residue; and substituting one ormore of the amino acid residues located in the P1′-P6’ region of thefurin-cleavage motif with one or more bulky and hydrophobic amino acidresidues

In certain embodiments, the disruption of the furin-cleavage motifcomprises a deletion, insertion, inversion, and/or mutation of at leastone amino acid residue within the furin-cleavage motif. In certainembodiments, a protease-cleavage resistant, Shiga toxin effectorpolypeptide of the present invention may comprise a disruption of theamino acid sequence natively positioned at 248-251 of the A Subunit ofShiga-like toxin 1 (SEQ ID NO:1), Shiga toxin (SEQ ID NO:2), or anotherShiga toxin 1 A Subunit variant (e.g. SEQ ID NOs: 4-6) or at 247-250 ofthe A Subunit of Shiga-like toxin 2 (SEQ ID NO:3) or a Shiga-like toxin2 variant (e.g. SEQ ID NOs: 7-18) or the equivalent position in aconserved Shiga toxin effector polypeptide and/or non-native Shiga toxineffector polypeptide sequence. In certain further embodiments,protease-cleavage resistant, Shiga toxin effector polypeptides comprisea disruption which comprises a deletion of at least one amino acidwithin the furin-cleavage motif. In certain further embodiments,protease-cleavage resistant, Shiga toxin effector polypeptides comprisea disruption which comprises an insertion of at least one amino acidwithin the protease-cleavage motif region. In certain furtherembodiments, the protease-cleavage resistant, Shiga toxin effectorpolypeptides comprise a disruption which comprises an inversion of aminoacids, wherein at least one inverted amino acid is within the proteasemotif region. In certain further embodiments, the protease-cleavageresistant, Shiga toxin effector polypeptides comprise a disruption whichcomprises a mutation, such as an amino acid substitution to anon-standard amino acid or an amino acid with a chemically modified sidechain. Examples of single amino acid substitutions are provided in theExamples below.

In certain embodiments of the molecules of the present invention, thedisrupted furin-cleavage motif comprises the deletion of nine, ten,eleven or more of the carboxy-terminal amino acid residues within thefurin-cleavage motif. In these embodiments, the disrupted furin-cleavagemotif will not comprise a furin-cleavage site or a minimalfurin-cleavage motif. In other words, certain embodiments lack afurin-cleavage site at the carboxy-terminus of the A1 fragment region.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue deletion and an amino acid residuesubstitution as compared to a wild-type, Shiga toxin A Subunit. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more amino acid residue deletions and substitutionswithin the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., forStxA and SLT-1A (and other Shiga toxin 1 A Subunit variants) derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue R248 substituted with any non-positively charged, amino acidresidue and/or R251 substituted with any non-positively charged, aminoacid residue; and for SLT-2A (and other Shiga-like toxin A Subunit 2variants) derived Shiga toxin effector polypeptides, the nativelypositioned amino acid residue R/Y247 substituted with any non-positivelycharged, amino acid residue and/or R250 substituted with anynon-positively charged, amino acid residue.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion and an amino acid residue substitution aswell as a carboxy-terminal truncation as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residue deletionsand substitutions within the minimal furin-cleavage site R/Y-x-x-R, suchas, e.g., for StxA and SLT-1A (and other Shiga toxin 1 A Subunitvariants) derived Shiga toxin effector polypeptides, the nativelypositioned amino acid residue R248 substituted with any non-positivelycharged, amino acid residue and/or R251 substituted with anynon-positively charged, amino acid residue; and for SLT-2A (and otherShiga-like toxin A Subunit 2 variants) derived Shiga toxin effectorpolypeptides, the natively positioned amino acid residue R/Y247substituted with any non-positively charged, amino acid residue and/orR250 substituted with any non-positively charged, amino acid residue.

In certain further embodiments, the disrupted furin-cleavage motifcomprises both an amino acid substitution within the minimalfurin-cleavage site R/Y-x-x-R and a carboxy-terminal truncation ascompared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxAand SLT-1A (and other Shiga toxin 1 A Subunit variants) derived Shigatoxin effector polypeptides, truncations ending at the natively aminoacid position 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, or greater and comprising the natively positionedamino acid residue R248 and/or R251 substituted with any non-positivelycharged, amino acid residue where appropriate; and for SLT-2A (and otherShiga-like toxin A Subunit 2 variants) derived Shiga toxin effectorpolypeptides, truncations ending at the natively amino acid position248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, or greater and comprising the natively positioned amino acidresidue R/Y247 and/or R250 substituted with any non-positively charged,amino acid residue where appropriate. In certain further embodiments,the furin-cleavage motif is disrupted by a carboxy-terminal truncationof the Shiga toxin A1 fragment region as compared to thecarboxy-terminus of a wild-type Shiga toxin A Subunit; wherein thecarboxy-terminal truncation ends at the natively amino acid position249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, or greater; and wherein the disrupted furin-cleavage motifcomprises the natively positioned amino acid residue R248 and/or R251 ofthe A Subunit of Shiga-like toxin 1 (SEQ ID NO:1), Shiga toxin (SEQ IDNO:2) or another Shiga toxin 1 A Subunit variant (see e.g. SEQ ID NOs:4-6), or the natively positioned amino acid residue R/Y247 and/or R250of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3) or a Shiga-liketoxin 2 A Subunit effector polypeptide variant (e.g. SEQ ID NOs: 7-18)substituted with an alanine residue. In certain further embodiments, thefurin-cleavage motif is disrupted by a carboxy-terminal truncation ofthe Shiga toxin A1 fragment region as compared to the carboxy-terminusof a wild-type Shiga toxin A Subunit; wherein the carboxy-terminaltruncation ends at the natively amino acid position 250, 249, 248, 247,or less. In certain embodiments, the carboxy-terminal truncation ends atthe natively amino acid position 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, or 261. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position250. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 251. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position252. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 253. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position254. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 255. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position256. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 257. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position258. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 259. In certain embodiments, thecarboxy-terminal truncation ends at the natively amino acid position260. In certain embodiments, the carboxy-terminal truncation ends at thenatively amino acid position 261.

In certain embodiments, the disrupted furin-cleavage motif comprises aninsertion of one or more amino acid residues as compared to a wild-type,Shiga toxin A Subunit as long as the inserted amino residue(s) does notcreate a de novo furin-cleavage site. In certain embodiments, theinsertion of one or more amino acid residues disrupts the naturalspacing between the arginine residues in the minimal, furin-cleavagesite R/Y-x-x-R, such as, e.g., StxA and SLT-1A (and other Shiga toxin 1A Subunit variants) derived polypeptides comprising an insertion of oneor more amino acid residues at 249 or 250 and thus between R248 andR251; or SLT-2A derived polypeptides (and other Shiga-like toxin 2 ASubunit variants) comprising an insertion of one or more amino acidresidues at 248 or 249 and thus between R/Y247 and R250.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue insertion and a carboxy-terminal truncationas compared to a wild-type, Shiga toxin A Subunit. In certainembodiments, the disrupted furin-cleavage motif comprises both an aminoacid residue insertion and an amino acid residue substitution ascompared to a wild-type, Shiga toxin A Subunit. In certain embodiments,the disrupted furin-cleavage motif comprises both an amino acid residueinsertion and an amino acid residue deletion as compared to a wild-type,Shiga toxin A Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, an amino acid residue insertion, and anamino acid residue substitution as compared to a wild-type, Shiga toxinA Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, insertion, substitution, andcarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit.

In certain embodiments, the Shiga toxin effector polypeptide comprisinga disrupted furin-cleavage motif is directly fused by a peptide bond toa molecular moiety comprising an amino acid, peptide, and/or polypeptidewherein the fused structure involves a single, continuous polypeptide.In these fusion embodiments, the amino acid sequence following thedisrupted furin-cleavage motif should not create a de novo,furin-cleavage site at the fusion junction.

Any of the above protease-cleavage resistant, Shiga toxin effectorpolypeptide sub-regions and/or disrupted furin-cleavage motifs may beused alone or in combination with each individual embodiment of thepresent invention, including methods of the present invention.

3. T-Cell Hyper-Immunized, Shiga Toxin A Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted epitope-peptide anda Shiga toxin A1 fragment derived region. In certain furtherembodiments, the epitope-peptide is a heterologous, T-cellepitope-peptide, such as, e.g., an epitope considered heterologous toShiga toxin A Subunits. In certain further embodiments, the Shiga toxineffector polypeptide of the present invention comprises an embedded orinserted epitope-peptide within the Shiga toxin A1 fragment region. Incertain further embodiments, the epitope-peptide is a CD8+ T-cellepitope. In certain further embodiments, the CD8+ T-cell epitope-peptidehas a binding affinity to a MHC class I molecule characterized by adissociation constant (K_(D)) of 10⁻⁴ molar or less and/or the resultingMHC class I-epitope-peptide complex has a binding affinity to a T-cellreceptor (TCR) characterized by a dissociation constant (K_(D)) of 10⁻⁴molar or less.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted, heterologous,T-cell epitope, such as, e.g., a human CD8+ T-cell epitope. In certainfurther embodiments, the heterologous, T-cell epitope is embedded orinserted so as to disrupt an endogenous epitope or epitope region (e.g.a B-cell epitope and/or CD4+ T-cell epitope) identifiable in a naturallyoccurring Shiga toxin polypeptide or parental Shiga toxin effectorpolypeptide from which the Shiga toxin effector polypeptide of thepresent invention is derived. For example, the Shiga toxin effectorpolypeptide of the present invention may comprise an embedded orinserted, heterologous, CD8+ T-cell epitope which disrupts anendogenous, B-cell and/or CD4+ T-cell epitope region within the Shigatoxin A1 fragment derived region.

For certain embodiments of the present invention, the Shiga toxineffector polypeptide (and any cell-targeting molecule comprising it) isCD8+ T-cell hyper-immunized, such as, e.g., as compared to a wild-typeShiga toxin polypeptide. The CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides of the present invention each comprise an embeddedor inserted T-cell epitope-peptide. Hyper-immunized, Shiga toxineffector polypeptides can be created from Shiga toxin effectorpolypeptides and/or Shiga toxin A Subunit polypeptides, whethernaturally occurring or not, using a method described herein, describedin WO 2015/113005, and/or known to the skilled worker, wherein theresulting molecule still retains one or more Shiga toxin A Subunitfunctions.

For purposes of the claimed invention, a T-cell epitope is a molecularstructure which is comprised by an antigenic peptide and can berepresented by a linear, amino acid sequence. Commonly, T-cell epitopesare peptides of sizes of eight to eleven amino acid residues (TownsendA, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however, certain T-cellepitope-peptides have lengths that are smaller than eight or larger thaneleven amino acids long (see e.g. Livingstone A, Fathman C, Annu RevImmunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34: 2510-9(2004)). In certain embodiments, the embedded or inserted epitope is atleast seven amino acid residues in length. In certain embodiments, theembedded or inserted epitope is bound by a TCR with a binding affinitycharacterized by a K_(D) less than 10 mM (e.g. 1-100 µM) as calculatedusing the formula in Stone J et al., Immunology 126: 165-76 (2009).However, it should be noted that the binding affinity within a givenrange between the MHC-epitope and TCR may not correlate withantigenicity and/or immunogenicity (see e.g. Al-Ramadi B et al., JImmunol 155: 662-73 (1995)), such as due to factors like MHC-peptide-TCRcomplex stability, MHC-peptide density and MHC-independent functions ofTCR cofactors such as CD8 (Baker B et al., Immunity 13: 475-84 (2000);Hornell T et al., J Immunol 170: 4506-14 (2003); Woolridge L et al., JImmunol 171: 6650-60 (2003)).

A heterologous, T-cell epitope is an epitope not already present in awild-type Shiga toxin A Subunit; a naturally occurring Shiga toxin ASubunit; and/or a parental, Shiga toxin effector polypeptide used as asource polypeptide for modification by a method described herein,described in WO 2015/113005, and/or known to the skilled worker.

A heterologous, T-cell epitope-peptide may be incorporated into a sourcepolypeptide via numerous methods known to the skilled worker, including,e.g., the processes of creating one or more amino acid substitutionswithin the source polypeptide, fusing one or more amino acids to thesource polypeptide, inserting one or more amino acids into the sourcepolypeptide, linking a peptide to the source polypeptide, and/or acombination of the aforementioned processes. The result of such a methodis the creation of a modified variant of the source polypeptide whichcomprises one or more embedded or inserted, heterologous, T-cellepitope-peptides.

T-cell epitopes may be chosen or derived from a number of sourcemolecules for use in the present invention. T-cell epitopes may becreated or derived from various naturally occurring proteins. T-cellepitopes may be created or derived from various naturally occurringproteins foreign to mammals, such as, e.g., proteins of microorganisms.T-cell epitopes may be created or derived from mutated human proteinsand/or human proteins aberrantly expressed by malignant human cells.T-cell epitopes may be synthetically created or derived from syntheticmolecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); DelVal M et al., J Virol 65: 3641-6 (1991); Appella E et al., Biomed PeptProteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116:2071-80 (2010)).

Although any T-cell epitope-peptide is contemplated as being used as aheterologous, T-cell epitope of the present invention, certain epitopesmay be selected based on desirable properties. One objective of thepresent invention is to create CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides for administration to vertebrates, meaning thatthe heterologous, T-cell epitope is highly immunogenic and can elicitrobust immune responses in vivo when displayed complexed with a MHCclass I molecule on the surface of a cell. In certain embodiments, theShiga toxin effector polypeptide of the present invention comprises oneor more, embedded or inserted, heterologous, T-cell epitopes which areCD8+ T-cell epitopes. A Shiga toxin effector polypeptide of the presentinvention that comprises a heterologous, CD8+ T-cell epitope isconsidered a CD8+ T-cell hyper-immunized, Shiga toxin effectorpolypeptide.

T-cell epitope components of the present invention may be chosen orderived from a number of source molecules already known to be capable ofeliciting a vertebrate immune response. T-cell epitopes may be derivedfrom various naturally occurring proteins foreign to vertebrates, suchas, e.g., proteins of pathogenic microorganisms and non-self, cancerantigens. In particular, infectious microorganisms may contain numerousproteins with known antigenic and/or immunogenic properties. Further,infectious microorganisms may contain numerous proteins with knownantigenic and/or immunogenic sub-regions or epitopes.

For example, the proteins of intracellular pathogens with mammalianhosts are sources for T-cell epitopes. There are numerous intracellularpathogens, such as viruses, bacteria, fungi, and single-cell eukaryotes,with well-studied antigenic proteins or peptides. T-cell epitopes can beselected or identified from human viruses or other intracellularpathogens, such as, e.g., bacteria like mycobacterium, fungi liketoxoplasmae, and protists like trypanosomes.

For example, there are many immunogenic, viral peptide components ofviral proteins from viruses that are infectious to humans. Numerous,human T-cell epitopes have been mapped to peptides within proteins frominfluenza A viruses, such as peptides in the proteins HA glycoproteinsFE17, S139/1, CH65, C05, hemagglutinin 1 (HA1), hemagglutinin 2 (HA2),nonstructural protein 1 and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1and M2), nucleoprotein (NP), neuraminidase (NA)), and many of thesepeptides have been shown to elicit human immune responses, such as byusing ex vivo assay. Similarly, numerous, human T-cell epitopes havebeen mapped to peptide components of proteins from humancytomegaloviruses (HCMV), such as peptides in the proteins pp65 (UL83),UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B, tegumentproteins, and many of these peptides have been shown to elicit humanimmune responses, such as by using ex vivo assays.

Another example is there are many immunogenic, cancer antigens inhumans. The CD8+ T-cell epitopes of cancer and/or tumor cell antigenscan be identified by the skilled worker using techniques known in theart, such as, e.g., differential genomics, differential proteomics,immunoproteomics, prediction then validation, and genetic approacheslike reverse-genetic transfection (see e.g., Admon A et al., Mol CellProteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3:193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).There are many antigenic and/or immunogenic T-cell epitopes alreadyidentified or predicted to occur in human cancer and/or tumor cells. Forexample, T-cell epitopes have been predicted in human proteins commonlymutated or overexpressed in neoplastic cells, such as, e.g., ALK, CEA,N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER2/neu, MAGE,Melan-A/MART-1, MUC-1, p53, and TRAG-3 (see e.g., van der Bruggen P etal., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180:347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Yet al., J Exp Med 183: 1173 (1996); Skipper J et al., J Exp Med 183: 527(1996); Brossart P et al., 93: 4309-17 (1999); Kawashima I et al.,Cancer Res 59: 431-5 (1999); Papadopoulos K et al., Clin Cancer Res 5:2089-93 (1999); Zhu B et al., Clin Cancer Res 9: 1850-7 (2003); Li B etal., Clin Exp Immunol 140: 310-9 (2005); Ait-Tahar K et al., IntJ Cancer118: 688-95 (2006); Akiyama Y et al., Cancer Immunol Immunother 61:2311-9 (2012)). In addition, synthetic variants of T-cell epitopes fromhuman cancer cells have been created (see e.g., Lazoura E,Apostolopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C etal., J Med Chem 50: 1598-609 (2007)).

While any T-cell epitope may be used in the polypeptides and moleculesof the present invention, certain T-cell epitopes may be preferred basedon their known and/or empirically determined characteristics. Forexample, in many species, the MHC alleles in its genome encode multipleMHC-I molecular variants. Because MHC class I protein polymorphisms canaffect antigen-MHC class I complex recognition by CD8+ T-cells, T-cellepitopes may be chosen for use in the present invention based onknowledge about certain MHC class I polymorphisms and/or the ability ofcertain antigen-MHC class I complexes to be recognized by T-cells havingdifferent genotypes.

There are well-defined peptide-epitopes that are known to beimmunogenic, MHC class I restricted, and/or matched with a specifichuman leukocyte antigen (HLA) variant(s). For applications in humans orinvolving human target cells, HLA-class I-restricted epitopes can beselected or identified by the skilled worker using standard techniquesknown in the art. The ability of peptides to bind to human MHC class Imolecules can be used to predict the immunogenic potential of putativeT-cell epitopes. The ability of peptides to bind to human MHC class Imolecules can be scored using software tools. T-cell epitopes may bechosen for use as a heterologous, T-cell epitope component of thepresent invention based on the peptide selectivity of the HLA variantsencoded by the alleles more prevalent in certain human populations. Forexample, the human population is polymorphic for the alpha chain of MHCclass I molecules due to the varied alleles of the HLA genes fromindividual to individual. In certain T-cell epitopes may be moreefficiently presented by a specific HLA molecule, such as, e.g., thecommonly occurring HLA variants encoded by the HLA-A allele groupsHLA-A2 and HLA-A3.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, multiple factors may be consideredthat can influence epitope generation and transport to receptive MHCclass I molecules, such as, e.g., the presence and epitope specificityof the following factors in the target cell: proteasome, ERAAP/ERAP1,tapasin, and TAPs.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, epitope may be selected which bestmatch the MHC class I molecules present in the cell-type or cellpopulations to be targeted. Different MHC class I molecules exhibitpreferential binding to particular peptide sequences, and particularpeptide-MHC class I variant complexes are specifically recognized by thet-cell receptors (TCRs) of effector T-cells. The skilled worker can useknowledge about MHC class I molecule specificities and TCR specificitiesto optimize the selection of heterologous, T-cell epitopes used in thepresent invention.

In addition, multiple, immunogenic, T-cell epitopes for MHC class Ipresentation may be embedded in the same Shiga toxin effectorpolypeptide of the present invention, such as, e.g., for use in thetargeted delivery of a plurality of T-cell epitopes simultaneously.

Any of the protease-cleavage resistant, Shiga toxin effector polypeptidesub-regions and/or disrupted furin-cleavage motifs described herein maybe used alone or in combination with each individual embodiment of thepresent invention, including methods of the present invention.

C. Additional Exogenous Materials

In certain embodiments, the cell-targeting molecules of the presentinvention comprises an additional exogenous material. An “additionalexogenous material” as used herein refers to one or more atoms ormolecules, often not generally present in both Shiga toxins and nativetarget cells, where the cell-targeting molecule of the present inventioncan be used to specifically transport such material to the interior of acell. In one sense, the entire cell-targeting molecule of the inventionis an exogenous material which will enter the cell; thus, the“additional” exogenous materials are heterologous materials linked tobut other than the core cell-targeting molecule itself. Non-limitingexamples of additional exogenous materials are radionuclides, peptides,detection promoting agents, proteins, small molecule chemotherapeuticagents, and polynucleotides.

In certain embodiments of the cell-targeting molecules of the presentinvention, the additional exogenous material is one or moreradionuclides, such as, e.g., ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹¹¹In, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, and/or radioactive isotopes of lutetium.

In certain embodiments, the additional exogenous material comprises aproapoptotic peptide, polypeptide, or protein, such as, e.g., BCL-2,caspases (e.g. fragments of caspase-3 or caspase-6), cytochromes,granzyme B, apoptosis-inducing factor (AIF), BAX, tBid (truncated Bid),and proapoptotic fragments or derivatives thereof (see e.g., Ellerby Het al., Nat Med 5: 1032-8 (1999); Mai J et al., Cancer Res 61: 7709-12(2001); Jia L et al., Cancer Res 63: 3257-62 (2003); Liu Y et al., MolCancer Ther 2: 1341-50 (2003); Perea S et al., Cancer Res 64: 7127-9(2004); Xu Y et al., J Immunol 173: 61-7 (2004); Dälken B et al., CellDeath Differ 13: 576-85 (2006); Wang T et al., Cancer Res 67: 11830-9(2007); Kwon M et al., Mol Cancer Ther 7: 1514-22 (2008); Qiu X et al.,Mol Cancer Ther 7: 1890-9 (2008); Shan L et al., Cancer Biol Ther 11:1717-22 (2008); Wang F et al., Clin Cancer Res 16: 2284-94 (2010); Kim Jet al., J Virol 85: 1507-16 (2011)).

In certain embodiments, the additional exogenous material comprises aprotein or polypeptide comprising an enzyme. In certain otherembodiments, the additional exogenous material is a nucleic acid, suchas, e.g. a ribonucleic acid that functions as a small inhibiting RNA(siRNA) or microRNA (miRNA). In certain embodiments, the additionalexogenous material is an antigen, such as antigens derived frompathogens, bacterial proteins, viral proteins, proteins mutated incancer, proteins aberrantly expressed in cancer, or T-cell complementarydetermining regions. For example, exogenous materials include antigens,such as those characteristic of antigen-presenting cells infected bybacteria, and T-cell complementary determining regions capable offunctioning as exogenous antigens. Exogenous materials comprisingpolypeptides or proteins may optionally comprise one or more antigenswhether known or unknown to the skilled worker.

In certain embodiments of the cell-targeting molecules of the presentinvention, all heterologous antigens and/or epitopes associated with theShiga toxin effector polypeptide are arranged in the cell-targetingmolecule amino-terminal to the carboxy-terminus of the Shiga toxin A1fragment region of the Shiga toxin effector polypeptide. In certainfurther embodiments, all heterologous antigens and/or epitopesassociated with the Shiga toxin effector polypeptide are associated,either directly or indirectly, with the Shiga toxin effector polypeptideat a position amino-terminal to the carboxy-terminus of the Shiga toxinAl fragment region of the Shiga toxin effector polypeptide. In certainfurther embodiments, all additional exogenous material(s) that is anantigen is arranged amino-terminal to the Shiga toxin effectorpolypeptide, such as, e.g., fused directly or indirectly to the aminoterminus of the Shiga toxin effector polypeptide.

In certain embodiments of the cell-targeting molecules of the presentinvention, the additional exogenous material is a cytotoxic agent, suchas, e.g., a small molecule chemotherapeutic agent, antineoplastic agent,cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomeraseinhibitor, and/or tubulin inhibitor. Non-limiting examples of cytotoxicagents suitable for use with the present invention include aziridines,cisplatins, tetrazines, procarbazine, hexamethylmelamine, vincaalkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone,teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin,amanitin, amatoxins, bleomycin, centanamycin (indolecarboxamide),plicamycin, mitomycin, daunorubicin, epirubicin, idarubicins,dolastatins, maytansines, maytansionoids, duromycin, docetaxel,duocarmycins, adriamycin, calicheamicin, auristatins,pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers (PBDs),carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C,paclitaxel, 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin,cisplatin, methotrexate, gemcitabine, aceglatone, acetogenins (e.g.bullatacin and bullatacinone), aclacinomysins, AG1478, AG1571,aldophosphamide glycoside, alkyl sulfonates (e.g., busulfan,improsulfan, and piposulfan), alkylating agents (e.g. thiotepa andcyclosphosphamide), aminolevulinic acid, aminopterin, amsacrine,ancitabine, anthramycin, arabinoside, azacitidine, azaserine, aziridines(e.g., benzodopa, carboquone, meturedopa, and uredopa), azauridine,bestrabucil, bisantrene, bisphosphonates (e.g. clodronate), bleomycins,bortezomib, bryostatin, cactinomycin, callystatin, carabicin,carminomycin, carmofur, carmustine, carzinophilin, CC-1065,chlorambucil, chloranbucil, chlornaphazine, chlorozotocin,chromomycinis, chromoprotein enediyne antibiotic chromophores, CPT-11,cryptophycins (e.g. cryptophycin 1 and cryptophycin 8),cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunomycin,defofamine, demecolcine, detorubicin, diaziquone,6-diazo-5-oxo-L-norleucine, dideoxyuridine, difluoromethylomithine(DMFO), doxifluridine, doxorubicins (e.g., morpholinodoxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin, anddeoxydoxorubicin), dynemicins, edatraxate, edatrexate, eleutherobins,elformithine, elliptinium acetate, enediyne antibiotics (e.g.calicheamicins), eniluracil, enocitabine, epirubicins, epothilone,esorubicins, esperamicins, estramustine, ethylenimines,2-ethylhydrazide, etoglucid, fludarabine, folic acid analogues (e.g.,denopterin, methotrexate, pteropterin, and trimetrexate), folic acidreplenishers (e.g. frolinic acid), fotemustine, fulvestrant, gacytosine,gallium nitrate, gefitinib, gemcitabine, hydroxyurea, ibandronate,ifosfamide, imatinib mesylate, erlotinib, fulvestrant, letrozole,PTK787/ZK 222584 (Novartis, Basel, CH), oxaliplatin, leucovorin,rapamycin, lapatinib, lonafamib, sorafenib, methylamelamines (e.g.,altretamine, triethy lenemelamine, triethy lenephosphoramide,triethylenethiophosphoramide and trimethylomelamine), pancratistatins,sarcodictyins, spongistatins, nitrogen mustards (e.g., chlorambucil,chlomaphazine, cyclophosphamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard), nitrosureas (e.g., carmustine,fotemustine, lomustine, nimustine, and ranimnustine), dynemicins,neocarzinostatin chromophores, anthramycin, detorubicin, epirubicins,marcellomycins, mitomycins (e.g. mitomycin C), mycophenolic acid,nogalamycins, olivomycins, peplomycins, potfiromycins, puromycins,quelamycins, rodorubicins, ubenimex, zinostatins, zorubicins, purineanalogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, andthioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine,6-azauridine, dideoxyuridine, doxifluridine, enocitabine, andfloxuridine), aceglatone, lentinan, lonidainine, maytansinoids (e.g.maytansins and ansamitocins), mitoguazone, mitoxantrone, mopidanmol,nitraerine, pentostatin, phenamet, pirarubicin, podophyllinic acid,2-ethylhydrazide, rhizoxin, sizofuran, spirogermanium, tenuazonic acid,triaziquone, 2,2’,2″trichlorotriethylamine, trichothecenes (e.g., T-2toxin, verracurin A, roridin A, and anguidine), urethan, vindesine,mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside,cyclophosphamide, toxoids (e.g. paclitaxel and doxetaxel),6-thioguanine, mercaptopurine, platinum, platinum analogs (e.g.cisplatin and carboplatin), etoposide (VP-16), mitoxantrone,vinorelbine, novantrone, daunomycin, xeloda, topoisomerase inhibitor RFS2000, retinoids (e.g. retinoic acid), capecitabine, lomustine,losoxantrone, mercaptopurines, nimustine, nitraerine, rapamycin,razoxane, roridin A, spongistatins, streptonigrins, streptozocins,sutent, T-2 toxin, thiamiprine, thiotepa, toxoids (e.g. paclitaxel anddoxetaxel), tubercidins, verracurin A, vinblastine, vincristine, andstructural analogs of any of the aforementioned (e.g. syntheticanalogs), and/or derivatives of any of the aforementioned (see e.g.,Lindell T et al., Science 170: 447-9 (1970); Remillard S et al., Science189: 1002-5 (1975); Ravry M et al., Am J Clin Oncol 8: 148-50 (1985);Ravry M et al., Cancer Treat Rep 69: 1457-8 (1985); Sternberg C et al.,Cancer 64: 2448-58 (1989); Bai R et al., Biochem Pharmacol 39: 1941-9(1990); Boger D, Johnson D, Proc Natl Acad Sci USA 92: 3642-9 (1995);Beck J et al., Leuk Lymphoma 41: 117-24 (2001); Cassady J et al., ChemPharm Bull (Tokyo) 52: 1-26 (2004); Sapra P et al., Clin Cancer Res 11:5257-64 (2005); Okeley N et al., Clinc Cancer Res 16: 888-97 (2010);Oroudjev E et al., Mol Cancer Ther 9: 2700-13 (2010); Ellestad G,Chirality 23: 660-71 (2011); Kantarjian H et al., Lancet Oncol 13:403-11 (2012); Moldenhauer G et al., J Natl Cancer Inst 104: 622-34(2012); Meulendijks D et al., Invest New Drugs 34: 119-28 (2016)).

D. Structure-Function Relationships of Cell-Targeting Molecules of theInvention

For certain embodiments of the cell-targeting molecules of the presentinvention, there specific structure-function relationships that havebeen observed, such as, e.g., component relative orientation effects oncytotoxic potency; furin-cleavage sensitivity effects on in vivotolerability at certain dosages; furin-cleavage sensitivity effects onin vitro stability; furin-cleavage sensitivity effects on in vivohalf-life; and furin-cleavage sensitivity effects on in vivo,non-specific toxicity in multicellular organisms.

In certain embodiments of the cell-targeting molecules of the presentinvention, the specific order or orientation of the Shiga toxin effectorpolypeptide region and binding region is fixed such that the bindingregion is located within the cell-targeting molecules more proximal tothe carboxy-terminus of the Shiga toxin effector polypeptide region thanto the amino-terminus of the Shiga toxin effector polypeptide region. Incertain embodiments of the cell-targeting molecules of the presentinvention, the arrangement of the Shiga toxin effector polypeptideregion within the cell-targeting molecule is limited to being at and/orproximal to the amino-terminus of a polypeptide component of thecell-targeting molecule (see FIG. 1 ). For example, certain embodimentsof the cell-targeting molecule of the present invention comprise 1) abinding region oriented within the cell-targeting molecule at a positioncarboxy-terminal to the Shiga toxin effector polypeptide region, 2) abinding region associated with the Shiga toxin effector polypeptideregion at a position distal from the amino-terminus of the Shiga toxineffector polypeptide region (e.g. distances of 50, 100, 200, or 250amino acid residues or greater), 3) a binding region not stericallycovering the amino-terminus of the Shiga toxin effector polypeptideregion, and/or 4) a binding region not sterically hindering astructure(s) near the amino-terminus of the Shiga toxin effectorpolypeptide region (see e.g. Figure 1; WO 2015/138452). In certainfurther embodiments, the cell-targeting molecules of the presentinvention are capable of exhibiting more optimal cytotoxic potency, suchas, e.g., exhibiting a CD₅₀ value which is 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or higher than a relatedcell-targeting reference molecule comprising the same Shiga toxin ASubunit effector polypeptide region(s) and binding region(s), whereinthe binding region is 1) amino-terminal to the Shiga toxin A Subuniteffector polypeptide region, 2) associated with the Shiga toxin effectorpolypeptide region at a position proximal to the amino-terminus of theShiga toxin effector polypeptide region (e.g. distances of less than 50,40, 30, 20, or 10 amino acid residues or less), 3) not stericallycovering the amino-terminus of the Shiga toxin effector polypeptideregion, and/or 4) not sterically hindering a structure(s) near theamino-terminus of the Shiga toxin effector polypeptide region (see e.g.Figure 1; WO 2015/138452).

In certain embodiments, the Shiga toxin A Subunit effector polypeptideof the present invention comprises a Shiga toxin A1 fragment derivedregion comprising a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment derived region (such asa disrupted furin-cleavage site located at the carboxy-terminus of aShiga toxin A1 fragment region) (see e.g. Figure 1; WO 2015/191764). Incertain further embodiments, the Shiga toxin effector polypeptide ismore furin-cleavage resistant as compared to a related referencemolecule, such as, e.g., a molecule comprising a wild-type, Shiga toxinA Subunit or Shiga toxin A1 fragment (see e.g. WO 2015/191764). Incertain further embodiments, the Shiga toxin effector polypeptide of thepresent invention exhibits a reduction in furin-cleavage reproduciblyobserved to be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, orless (including 100% for no cleavage) than the furin-cleavage of areference molecule observed in the same assay under the same conditions.In certain further embodiments, the Shiga toxin effector polypeptide ismore cleavage resistant to a protease other than furin as compared to arelated reference molecule, such as, e.g., a molecule comprising awild-type, Shiga toxin A Subunit or Shiga toxin A1 fragment.

Certain cell-targeting molecules of the present invention exhibitcytotoxic potencies within 100-fold, 20-fold, 10-fold, 5-fold, or lessthan a reference molecule comprising a wild-type Shiga toxin effectorpolypeptide region despite the lack of any compensatory structuralfeature for the disrupted furin-cleavage motif in the Shiga toxineffector polypeptide. For cell-targeting molecules comprising Shigatoxin A Subunit derived regions which do not maintain the furin cleavageevent, i.e. molecules comprising Shiga toxin A Subunit derivedcomponents which are not cleaved by furin inside target cells, onealternative for preserving maximal cytotoxicity is compensation.Compensation for the lack of furin cleavage of a Shiga toxin A Subunitregion in cytotoxic molecule might be accomplished by presenting theShiga toxin A Subunit region in a “pre-processed” form. For example, acell-targeting molecule comprising a Shiga toxin A Subunit region may beconstructed such that the carboxy-terminus of the Shiga toxin A Subunitderived polypeptide is 1) proximal to a carboxy-terminus of the moleculeand 2) matches or resembles a native Shiga toxin A1 fragment aftercleavage by furin (see WO 2015/191764). Such compensation is notrequired in certain cell-targeting molecules of the present invention,rather it is intentionally avoided in order to provide one or morefunction(s), such as, e.g., improved in vivo tolerability at certaindosages; increased in vitro stability; increased in vivo half-life;and/or reduced in vivo, non-specific toxicity in multicellularorganisms. For certain embodiments, these beneficial function(s) arepresent without any significant reduction in cytotoxic potency of thecell-targeting molecule of the present invention as compared to areference molecule comprising a wild-type Shiga toxin effectorpolypeptide.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin A Subunit effector polypeptidecomprising a Shiga toxin A1 fragment derived region comprising adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region (such as a disrupted furin-cleavagesite located at the carboxy-terminus of a Shiga toxin A1 fragmentregion) (see e.g. Figure 1; WO 2015/191764) but do not comprise anycompensatory protease cleavage site proximal to the carboxy-terminus ofthe Shiga toxin A1 fragment derived region and/or oriented between theShiga toxin effector polypeptide and a relatively large, molecule moiety(e.g. a binding region of a size greater than 4.5 kDa, 6, kDa, 9 kDa, 12kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, or 50 kDa). Incertain further embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin effector polypeptide which is morefurin-cleavage resistant as compared to a related reference molecule,such as, e.g., a molecule comprising a wild-type, Shiga toxin A Subunitor Shiga toxin A1 fragment (see e.g. WO 2015/191764). In certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits a reduction in furin-cleavage of 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 97%, 98%, 99%, or 100% less than the furin-cleavage of areference molecule observed in the same assay under the same conditionswhile the cell-targeting molecule exhibits a cytotoxic potency within100-fold, 20-fold, 10-fold, 5-fold, or less than a reference moleculecomprising a wild-type Shiga toxin effector polypeptide region. Incertain further embodiments, the cell-targeting molecule of the presentinvention exhibits an improvement in in vivo tolerability as compared toa related reference molecule comprising a Shiga toxin effectorpolypeptide having a wild-type furin cleavage motif and/or wild-typefurin cleavage site at the carboxy-terminus of its Shiga toxin A1fragment region (see e.g. WO 2015/191764). For example, an increase inin vivo tolerability may be determined by comparing measurements ofmortality, signs of morbidity, and/or certain clinical signs in groupsof laboratory animals administered different molecules at the samedosages (see e.g. Examples, infra; WO 2015/191764; WO 2016/196344).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin A Subunit effector polypeptidecomprising a Shiga toxin A1 fragment derived region comprising adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region (such as a disrupted furin-cleavagesite located at the carboxy-terminus of a Shiga toxin A1 fragmentderived region) (see e.g. Figure 1; WO 2015/191764). For certain furtherembodiments, the cell-targeting molecule of the present invention thatcomprise a cytotoxic component, the cell-targeting molecule exhibitsreduced non-specific toxicity as compared to more protease-cleavagesensitive variants, which have greater propensity to break apart andthereby release the cytotoxic component from the binding region,especially when administered to living materials, such as, e.g., apopulation of cells, a tissue, and/or an organism. Furthermore, certainprotease-cleavage resistant, cell-targeting molecules of the presentinvention may exhibit increased, in vivo, half-lives afteradministration to living materials (e.g., certain chordates) as comparedto more protease-cleavage sensitive variants based on theprotease-cleavage resistance conferred to the cell-targeting molecule bythe disrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region.

III. Linkages Connecting Components of the Invention and/or TheirSubcomponents

Individual cell-targeting binding regions, Shiga toxin effectorpolypeptides, and/or components of the cell-targeting molecules presentinvention may be suitably linked to each other via one or more linkerswell known in the art and/or described herein. Individual polypeptidesubcomponents of the binding regions, e.g. heavy chain variable regions(V_(H)), light chain variable regions (V_(L)), CDR, and/or ABR regions,may be suitably linked to each other via one or more linkers well knownin the art and/or described herein. Proteinaceous components of theinvention, e.g., multi-chain binding regions, may be suitably linked toeach other or other polypeptide components of the invention via one ormore linkers well known in the art. Peptide components of the invention,e.g., KDEL family endoplasmic reticulum retention/retrieval signalmotifs, may be suitably linked to another component of the invention viaone or more linkers, such as a proteinaceous linker, which are wellknown in the art.

Suitable linkers are generally those which allow each polypeptidecomponent of the present invention to fold with a three-dimensionalstructure very similar to the polypeptide components producedindividually without any linker or other component. Suitable linkersinclude single amino acids, peptides, polypeptides, and linkers lackingany of the aforementioned, such as various non-proteinaceous carbonchains, whether branched or cyclic.

Suitable linkers may be proteinaceous and comprise one or more aminoacids, peptides, and/or polypeptides. Proteinaceous linkers are suitablefor both recombinant fusion proteins and chemically linked conjugates. Aproteinaceous linker typically has from about 2 to about 50 amino acidresidues, such as, e.g., from about 5 to about 30 or from about 6 toabout 25 amino acid residues. The length of the linker selected willdepend upon a variety of factors, such as, e.g., the desired property orproperties for which the linker is being selected. In certainembodiments, the linker is proteinaceous and is linked near the terminusof a protein component of the present invention, typically within about20 amino acids of the terminus.

Suitable linkers may be non-proteinaceous, such as, e.g. chemicallinkers. Various non-proteinaceous linkers known in the art may be usedto link cell-targeting binding regions to the Shiga toxin effectorpolypeptide components of the cell-targeting molecules of the presentinvention, such as linkers commonly used to conjugate immunoglobulinpolypeptides to heterologous polypeptides. For example, polypeptideregions may be linked using the functional side chains of their aminoacid residues and carbohydrate moieties such as, e.g., a carboxy, amine,sulfhydryl, carboxylic acid, carbonyl, hydroxyl, and/or cyclic ringgroup. For example, disulfide bonds and thioether bonds may be used tolink two or more polypeptides. In addition, non-natural amino acidresidues may be used with other functional side chains, such as ketonegroups. Examples of non-proteinaceous chemical linkers include but arenot limited to N-succinimidyl (4-iodoacetyl)-aminobenzoate,S-(N-succinimidyl) thioacetate (SATA),N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio) toluene (SMPT),N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC),sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio) toluene,sulfosuccinimidyl-6-(α-methyl-α-(pyridyldithiol)-toluamido) hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP), succinimidyl6(3(-(-2-pyridyldithio)-proprionamido) hexanoate, sulfosuccinimidyl6(3(-(-2-pyridyldithio)-propionamido) hexanoate, maleimidocaproyl (MC),maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB),3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), alpha-alkylderivatives, sulfoNHS-ATMBA (sulfosuccinimidylN-[3-(acetylthio)-3-methylbutyryl-beta-alanine]), sulfodichlorophenol,2-iminothiolane, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman’sreagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

Suitable linkers, whether proteinaceous or non-proteinaceous, mayinclude, e.g., protease sensitive, environmental redox potentialsensitive, pH sensitive, acid cleavable, photocleavable, and/or heatsensitive linkers.

Proteinaceous linkers may be chosen for incorporation into recombinantfusion cell-targeting molecules of the present invention. Forrecombinant fusion cell-targeting proteins of the invention, linkerstypically comprise about 2 to 50 amino acid residues, preferably about 5to 30 amino acid residues. Commonly, proteinaceous linkers comprise amajority of amino acid residues with polar, uncharged, and/or chargedresidues, such as, e.g., threonine, proline, glutamine, glycine, andalanine. Non-limiting examples of proteinaceous linkers includealanine-serine-glycine-glycine-proline-glutamate (ASGGPE (SEQ ID NO:158)), valine-methionine (VM), alanine-methionine (AM), AM(G₂ _(to)₄S)_(x)AM where G is glycine, S is serine, and x is an integer from 1 to10 (SEQ ID NO: 159).

Proteinaceous linkers may be selected based upon the properties desired.Proteinaceous linkers may be chosen by the skilled worker with specificfeatures in mind, such as to optimize one or more of the fusionmolecule’s folding, stability, expression, solubility, pharmacokineticproperties, pharmacodynamic properties, and/or the activity of the fuseddomains in the context of a fusion construct as compared to the activityof the same domain by itself. For example, proteinaceous linkers may beselected based on flexibility, rigidity, and/or cleavability. Theskilled worker may use databases and linker design software tools whenchoosing linkers. In certain linkers may be chosen to optimizeexpression. In certain linkers may be chosen to promote intermolecularinteractions between identical polypeptides or proteins to formhomomultimers or different polypeptides or proteins to formheteromultimers. For example, proteinaceous linkers may be selectedwhich allow for desired non-covalent interactions between polypeptidecomponents of the cell-targeting molecules of the invention, such as,e.g., interactions related to the formation dimers and other higherorder multimers.

Flexible proteinaceous linkers are often greater than 12 amino acidresidues long and rich in small, non-polar amino acid residues, polaramino acid residues, and/or hydrophilic amino acid residues, such as,e.g., glycines, serines, and threonines. Flexible proteinaceous linkersmay be chosen to increase the spatial separation between componentsand/or to allow for intramolecular interactions between components. Forexample, various “GS” linkers are known to the skilled worker and arecomposed of multiple glycines and/or one or more serines, sometimes inrepeating units, such as, e.g., G_(x)S)_(n) (SEQ ID NO: 160),(S_(x)G)_(n) (SEQ ID NO: 161), (GGGGS)_(n) (SEQ ID NO: 162), and(G)_(n), in which x is 1 to 6 and n is 1 to 30 (SEQ ID NO: 163).Non-limiting examples of flexible proteinaceous linkers includeGKSSGSGSESKS (SEQ ID NO: 164), EGKSSGSGSESKEF (SEQ ID NO: 165),GSTSGSGKSSEGKG (SEQ ID NO: 166), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 167),GSTSGSGKPGSGEGSTKG (SEQ ID NO:96), SRSSG (SEQ ID NO: 168), and SGSSC(SEQ ID NO: 169).

Rigid proteinaceous linkers are often stiff alpha-helical structures andrich in proline residues and/or one or more strategically placedprolines. Rigid linkers may be chosen to prevent intramolecularinteractions between linked components.

Suitable linkers may be chosen to allow for in vivo separation ofcomponents, such as, e.g., due to cleavage and/or environment-specificinstability. In vivo cleavable proteinaceous linkers are capable ofunlinking by proteolytic processing and/or reducing environments oftenat a specific site within an organism or inside a certain cell type. Invivo cleavable proteinaceous linkers often comprise protease sensitivemotifs and/or disulfide bonds formed by one or more cysteine pairs. Invivo cleavable proteinaceous linkers may be designed to be sensitive toproteases that exist only at certain locations in an organism,compartments within a cell, and/or become active only under certainphysiological or pathological conditions (such as, e.g., involvingproteases with abnormally high levels, proteases overexpressed atcertain disease sites, and proteases specifically expressed by apathogenic microorganism). For example, there are proteinaceous linkersknown in the art which are cleaved by proteases present onlyintracellularly, proteases present only within specific cell types, andproteases present only under pathological conditions like cancer orinflammation, such as, e.g., R-x-x-R motif and

AMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO: 170).

In certain embodiments of the cell-targeting molecules of the presentinvention, a linker may be used which comprises one or more proteasesensitive sites to provide for cleavage by a protease present within atarget cell. In certain embodiments of the cell-targeting molecules ofthe invention, a linker may be used which is not cleavable to reduceunwanted toxicity after administration to a vertebrate organism.

Suitable linkers may include, e.g., protease sensitive, environmentalredox potential sensitive, pH sensitive, acid cleavable, photocleavable,and/or heat sensitive linkers, whether proteinaceous ornon-proteinaceous (see e.g., Doronina S et al., Bioconjug Chem 17:114-24 (2003); Saito G et al., Adv Drug Deliv Rev 55: 199-215 (2003);Jeffrey S et al., J Med Chem 48: 1344-58 (2005); Sanderson R et al.,Clin Cancer Res 11: 843-52 (2005); Erickson H et al., Cancer Res 66:4426-33 (2006); Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)).Suitable cleavable linkers may include linkers comprising cleavablegroups which are known in the art.

Suitable linkers may include pH sensitive linkers. For example, certainsuitable linkers may be chosen for their instability in lower pHenvironments to provide for dissociation inside a subcellularcompartment of a target cell (see e.g., van Der Velden V et al., Blood97: 3197-204 (2001); Ulbrich K, Subr V, Adv Drug Deliv Rev 56: 1023-50(2004)). For example, linkers that comprise one or more trityl groups,derivatized trityl groups, bismaleimideothoxy propane groups, adipicacid dihydrazide groups, and/or acid labile transferrin groups, mayprovide for release of components of the cell-targeting molecules of theinvention, e.g. a polypeptide component, in environments with specificpH ranges. In certain linkers may be chosen which are cleaved in pHranges corresponding to physiological pH differences between tissues,such as, e.g., the pH of tumor tissue is lower than in healthy tissues.

Photocleavable linkers are linkers that are cleaved upon exposure toelectromagnetic radiation of certain wavelength ranges, such as light inthe visible range. Photocleavable linkers may be used to release acomponent of a cell-targeting molecule of the invention, e.g. apolypeptide component, upon exposure to light of certain wavelengths.Non-limiting examples of photocleavable linkers include a nitrobenzylgroup as a photocleavable protective group for cysteine,nitrobenzyloxycarbonyl chloride cross-linkers,hydroxypropylmethacrylamide copolymer, glycine copolymer, fluoresceincopolymer, and methylrhodamine copolymer. Photocleavable linkers mayhave particular uses in linking components to form cell-targetingmolecules of the invention designed for treating diseases, disorders,and conditions that can be exposed to light using fiber optics.

In certain embodiments of the cell-targeting molecules of the presentinvention, a cell-targeting binding region is linked to a Shiga toxineffector polypeptide of the present invention using any number of meansknown to the skilled worker, including both covalent and noncovalentlinkages.

In certain embodiments of the cell-targeting molecules of the presentinvention, the molecule comprises a binding region which is a scFv witha linker connecting a heavy chain variable (V_(H)) domain and a lightchain variable (V_(L)) domain. There are numerous linkers known in theart suitable for this purpose, such as, e.g., the 15-residue (Gly4Ser)₃peptide (SED ID NO:171). Suitable scFv linkers which may be used informing non-covalent multivalent structures include GGS, (SEQ IDNO:172), GGGGS (SEQ ID NO:94), GGGGSGGG (SEQ ID NO:173), GGSGGGG (SEQ IDNO:174), GSTSGGGSGGGSGGGGSS (SEQ ID NO:175), and GSTSGSGKPGSSEGSTKG (SEQID NO:176).

Suitable methods for linkage of the components of the cell-targetingmolecules of the present invention may be by any method presently knownin the art for accomplishing such, so long as the attachment does notsubstantially impede the binding capability of the cell-targetingbinding region, the cellular internalization of the Shiga toxin effectorpolypeptide component, and/or when appropriate the desired Shiga toxineffector function(s) as measured by an appropriate assay, includingassays described herein.

The components of the cell-targeting molecule, e.g. a Shiga toxin ASubunit effector polypeptide and/or immunoglobulin-type HER2-bindingregion, may be engineered to provide a suitable attachment moiety forthe linkage of additional components, e.g. an additional exogenousmaterial (see e.g. WO2018/106895).

For the purposes of the cell-targeting molecules of the presentinvention, the specific order or orientation is not fixed for thecomponents: the Shiga toxin effector polypeptide(s), the bindingregion(s), and any optional linker(s), in relation to each other or theentire cell-targeting molecule unless specifically noted. The componentsof the cell-targeting molecules of the present invention may be arrangedin any order provided that the desired activity(ies) of the bindingregion and Shiga toxin effector polypeptide are not eliminated.

IV. Examples of Structural Variations of the Shiga Toxin EffectorPolypeptides and Cell-Targeting Molecules of the Invention

In certain embodiments, a Shiga toxin effector polypeptide of thepresent invention may comprise, consist of, or consist essentially of atruncated Shiga toxin A Subunit. Truncations of Shiga toxin A Subunitsmight result in the deletion of an entire epitope(s) and/or epitoperegion(s), B-cell epitopes, CD4+ T-cell epitopes, and/or furin-cleavagesites without affecting Shiga toxin effector functions, such as, e.g.,catalytic activity and cytotoxicity. The smallest Shiga toxin A Subunitfragment shown to exhibit full enzymatic activity was a polypeptidecomposed of residues 1-239 of Slt1A (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). The smallest Shiga toxin A Subunit fragment shown toexhibit significant enzymatic activity was a polypeptide composed ofresidues 75-247 of StxA (Al-Jaufy A et al., Infect Immun 62: 956-60(1994)).

Although Shiga toxin effector polypeptides of the present invention maycommonly be smaller than the full-length Shiga toxin A Subunit, it ispreferred that the Shiga toxin effector polypeptide region of acell-targeting molecule of the present invention maintain thepolypeptide region from amino acid position 77 to 239 (SLT-1A (SEQ IDNO:1), StxA (SEQ ID NO:2), or Shiga toxin 1 A Subunit variants, e.g. SEQID NOs: 4-6) or the equivalent in other A Subunits of members of theShiga toxin family (e.g. 77 to 238 of SEQ ID NOs: 3 and 7-18)). Forexample, in certain embodiments of the molecules of the presentinvention, the Shiga toxin effector polypeptides of the presentinvention derived from SLT-1A may comprise, consist of, or consistessentially of amino acids 75 to 251 of SEQ ID NO: 1, 1 to 241 of SEQ IDNO:1, 1 to 251 of SEQ ID NO: 1, or amino acids 1 to 261 of SEQ ID NO: 1,further comprising relative to a wild-type Shiga toxin A Subunit atleast one amino acid residue which is mutated or has been deleted in anendogenous epitope and/or epitope region, and/or wherein there is adisrupted, furin-cleavage motif region at the carboxy-terminus of aShiga toxin A1 fragment derived region. Similarly, Shiga toxin effectorpolypeptide regions derived from Shiga toxin 1 A Subunit variants (suchas Stx1cA, Stx1dA, and Stx1eA) may comprise, consist essentially of, orconsist of amino acids 75 to 251 of SEQ ID NOs: 4-6, 1 to 241 of SEQ IDNOs: 4-6, or 1 to 251 of SEQ ID NOs: 4-6, further comprising relative toa wild-type Shiga toxin A Subunit at least one amino acid residue whichis mutated or has been deleted in an endogenous epitope and/or epitoperegion, and/or wherein there is a disrupted, furin-cleavage motif regionat the carboxy-terminus of a Shiga toxin A1 fragment derived region.Additionally, Shiga toxin effector polypeptide regions derived fromSLT-2 may comprise, consist of, or consist essentially of amino acids 75to 251 of SEQ ID NO:3, 1 to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3,or amino acids 1 to 261 of SEQ ID NO:3, further comprising relative to awild-type Shiga toxin A Subunit at least one amino acid residue which ismutated or has been deleted in an endogenous epitope and/or epitoperegion, and/or wherein there is a disrupted, furin-cleavage motif regionat the carboxy-terminus of a Shiga toxin A1 fragment derived region.Likewise, Shiga toxin effector polypeptide regions derived fromShiga-like toxin 2 A Subunit variants (such as Stx2cA variant 1, Stx2cAvariant 2, Stx2cA variant 3, Stx2cA variant 4, Stx2cA variant 5, Stx2cAvariant 6, Stx2dA variant 1, Stx2dA variant 2, Stx2dA variant 3, Stx2eAvariant 1, Stx2eA variant 2, and Stx2fA) may comprise, consistessentially of, or consist of amino acids 1 to 241 of SEQ ID NOs: 7-18,further comprising relative to a wild-type Shiga toxin A Subunit atleast one amino acid residue which is mutated or has been deleted in anendogenous epitope and/or epitope region, and/or wherein there is adisrupted, furin-cleavage motif region at the carboxy-terminus of aShiga toxin A1 fragment derived region.

In certain embodiments, the Shiga toxin effector polypeptide comprises,consists essentially of, or consists of: (i) amino acids 75 to 251 ofany one of SEQ ID NOs: 1-6; (ii) amino acids 1 to 241 of any one of SEQID NOs: 1-18; (iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6;and/or (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-3. Incertain embodiments, the Shiga toxin effector polypeptide comprises,consists essentially of, or consists of: (i) amino acids 75 to 251 ofany one of SEQ ID NOs: 1-6; (ii) amino acids 1 to 241 of any one of SEQID NOs: 1-18; (iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6;and/or (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-3, whereinrelative to a wild-type Shiga toxin A Subunit at least one amino acidresidue is mutated or has been deleted in an endogenous epitope and/orepitope region, and/or wherein there is a disrupted, furin-cleavagemotif region at the carboxy-terminus of a Shiga toxin A1 fragmentderived region.

The invention further provides variants of Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention,wherein the Shiga toxin effector polypeptide differs from a naturallyoccurring Shiga toxin A Subunit by only or up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by nomore than that which retains at least 85%, 90%, 95%, 99% or more aminoacid sequence identity). Thus, a molecule of the present inventionderived from an A Subunit of a member of the Shiga toxin family maycomprise additions, deletions, truncations, or other alterations fromthe original sequence as long as at least 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more amino acid sequence identity ismaintained to a naturally occurring Shiga toxin A Subunit and whereinrelative to a wild-type Shiga toxin A Subunit at least one amino acidresidue is mutated or has been deleted in an endogenous epitope and/orepitope region, and/or wherein there is a disrupted, furin-cleavagemotif region at the carboxy-terminus of a Shiga toxin A1 fragmentderived region.

Accordingly, in certain embodiments, the Shiga toxin effectorpolypeptide of a molecule of the present invention comprises, consistsof, or consists essentially of amino acid sequences having at least 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7%overall sequence identity to a naturally occurring Shiga toxin A Subunit(or a fragment thereof), such as SLT-1A (SEQ ID NO: 1), StxA (SEQ IDNO:2), Shiga toxin 1 A Subunit variants (e.g. SEQ ID NOs: 4-6), SLT-2A(SEQ ID NO:3), and/or Shiga-like toxin 2 A Subunit variants (e.g. SEQ IDNOs: 7-18), wherein relative to a wild-type Shiga toxin A Subunit atleast one amino acid residue is mutated or has been deleted in anendogenous epitope and/or epitope region, and/or wherein there is adisrupted, furin-cleavage motif region at the carboxy-terminus of aShiga toxin A1 fragment derived region. In certain embodiments, theShiga toxin effector polypeptide comprises, consists essentially of, orconsists of an amino acid sequence that is at least 85% identical (suchas at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or99.7% identical) to a wild-type Shiga toxin A Subunit amino acidsequence selected from: (i) amino acids 75 to 251 of any one of SEQ IDNOs: 1-6; (ii) amino acids 1 to 241 of any one of SEQ ID NOs: 1-18;(iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6; and (iv) aminoacids 1 to 261 of any one of SEQ ID NOs: 1-3. In certain embodiments,the Shiga toxin effector polypeptide comprises, consists essentially of,or consists of an amino acid sequence that is at least 85% identical(such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or 99.7% identical) to a wild-type Shiga toxin A Subunit aminoacid sequence selected from: (i) amino acids 75 to 251 of any one of SEQID NOs: 1-3; (ii) amino acids 1 to 241 of any one of SEQ ID NOs: 1-3;(iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-3; or (iv) aminoacids 1 to 261 of any one of SEQ ID NOs: 1-3. In certain embodiments,the Shiga toxin effector polypeptide comprises, consists essentially of,or consists of an amino acid sequence that is at least 85% identical(such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or 99.7% identical) to a wild-type Shiga toxin A Subunit aminoacid sequence selected from: (i) amino acids 75 to 251 of SEQ ID NO: 1;(ii) amino acids 1 to 241 of SEQ ID NO: 1; (iii) amino acids 1 to 251 ofSEQ ID NO: 1; or (iv) amino acids 1 to 261 of any one of SEQ ID NO: 1.In certain embodiments, the Shiga toxin effector polypeptide comprises,consists essentially of, or consists of an amino acid sequence that isat least 85% identical (such as at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5% or 99.7% identical) to a wild-type Shiga toxinA Subunit amino acid sequence selected from: (i) amino acids 75 to 251of SEQ ID NO:2; (ii) amino acids 1 to 241 of SEQ ID NO:2; (iii) aminoacids 1 to 251 of SEQ ID NO:2; or (iv) amino acids 1 to 261 of SEQ IDNO:2. In certain embodiments, the Shiga toxin effector polypeptidecomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% identical (such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% identical) to awild-type Shiga toxin A Subunit amino acid sequence selected from: (i)amino acids 75 to 251 of any one of SEQ ID NO:3; (ii) amino acids 1 to241 of SEQ ID NO:3; (iii) amino acids 1 to 251 of SEQ ID NO:3; or (iv)amino acids 1 to 261 of SEQ ID NO:3.

Optionally, either a full-length or a truncated version of the Shigatoxin A Subunit may comprise the Shiga toxin effector polypeptide regionof a molecule of the present invention, wherein the Shiga toxin derivedpolypeptide comprises one or more mutations (e.g. substitutions,deletions, insertions, or inversions) as compared to a naturallyoccurring Shiga toxin. It is preferred in certain embodiments of theinvention that the Shiga toxin effector polypeptides have sufficientsequence identity to a naturally occurring (or wild-type) Shiga toxin ASubunit to retain cytotoxicity after entry into a cell, either bywell-known methods of host cell transformation, transfection, infectionor induction, or by internalization mediated by a cell-targeting bindingregion linked with the Shiga toxin effector polypeptide. The mostcritical residues for enzymatic activity and/or cytotoxicity in theShiga toxin A Subunits have been mapped to the followingresidue-positions: asparagine-75, tyrosine-77, glutamate-167,arginine-170, and arginine-176 among others (Di R et al., Toxicon 57:525-39 (2011)). In any one of the embodiments of the invention, theShiga toxin effector polypeptides may preferably but not necessarilymaintain one or more conserved amino acids at positions, such as thosefound at positions 77, 167, 170, and 176 in StxA, SLT-1A, or theequivalent conserved position in other members of the Shiga toxin familywhich are typically required for cytotoxic activity. The capacity of acytotoxic molecule of the invention to cause cell death, e.g. itscytotoxicity, may be measured using any one or more of a number ofassays well known in the art.

A. Examples of De-Immunized, Shiga Toxin Effector Polypeptides

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention may consist essentially of atruncated Shiga toxin A Subunit having two or more mutations.Truncations of Shiga toxin A Subunits might result in the deletion of anentire epitope(s) and/or epitope region(s), B-cell epitopes, CD4+ T-cellepitopes, and/or furin-cleavage sites without affecting Shiga toxineffector functions, such as, e.g., catalytic activity and cytotoxicity.Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A to aminoacids 1-251 removes two predicted B-cell epitope regions, two predictedCD4 positive (CD4+) T-cell epitopes, and a predicted discontinuousB-cell epitope. These epitopes are also absent from the Shiga toxineffector polypeptides shown in SEQ ID NOs: 4-18. The Shiga toxin 1 ASubunit effector polypeptides shown in SEQ ID NOs: 4-6 relate tofragments of wild-type Shiga toxin A Subunit variants which have beentruncated at position 251, and the Shiga-like toxin 2 A Subunit effectorpolypeptides shown in SEQ ID NOs: 7-18 relate to fragments of theShiga-like toxin 2 A Subunit variants which have been truncated atposition 250. Truncating the amino-terminus of SLT-1A, StxA, or SLT-2Ato 75-293 removes at least three predicted B-cell epitope regions andthree predicted CD4+ T-cell epitopes. Truncating both amino- andcarboxy-terminals of SLT-1A, StxA, or SLT-2A to 75-251 deletes at leastfive predicted B-cell epitope regions, four putative CD4+ T-cellepitopes and one predicted discontinuous B-cell epitope.

In certain embodiments, a de-immunized, Shiga toxin effector polypeptideof the present invention may comprise, consist of, or consistessentially of a full-length or truncated Shiga toxin A Subunit with atleast one mutation (relative to a wild-type Shiga toxin polypeptide),e.g. deletion, insertion, inversion, or substitution, in a provided,endogenous, B-cell and/or CD4+ T-cell epitope region. In certainembodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises a mutation (relative toa wild-type Shiga toxin polypeptide) which includes a deletion of atleast one amino acid residue within the endogenous, B-cell and/or CD4+T-cell epitope region. In certain embodiments, the Shiga toxin effectorpolypeptide of the present invention comprises a disruption whichcomprises an insertion of at least one amino acid residue within theendogenous, B-cell and/or CD4+ T-cell epitope region. In certainembodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises an inversion of aminoacid residues, wherein at least one inverted amino acid residue iswithin the endogenous, B-cell and/or CD4+ T-cell epitope region. Incertain embodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises a mutation (relative toa wild-type Shiga toxin polypeptide), such as, e.g., an amino acidsubstitution, an amino acid substitution to a non-standard amino acid,and/or an amino acid residue with a chemically modified side chain.Non-limiting examples of de-immunized, Shiga toxin effector sub-regionssuitable for use in the present invention are described in WO2015/113005, WO 2015/113007 and WO 2015/191764. Numerous, non-limitingexamples of Shiga toxin effector polypeptides of the present inventionwhich comprise amino acid substitutions are provided in the Examples.

In other embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention comprises a truncated Shiga toxin A Subunitwhich is shorter than a full-length Shiga toxin A Subunit wherein atleast one amino acid residue is disrupted in a natively positioned,B-cell and/or CD4+ T-cell epitope region.

To create a de-immunized, Shiga toxin effector polypeptide, in principlemodifying any amino acid residue in a provided epitope region by variousmeans can result in a disruption of an epitope, such as, e.g., amodification which represents a deletion, insertion, inversion,rearrangement, substitution, and chemical modification of a side chainrelative to a wild-type Shiga toxin polypeptide. However, modifyingcertain amino acid residues and using certain amino acid modificationsare more likely to successfully reduce antigenicity and/orimmunogenicity while maintaining a certain level of a Shiga toxineffector function(s). For example, terminal truncations and internalamino acid substitutions are preferred because these types ofmodifications maintain the overall spacing of the amino acid residues ina Shiga toxin effector polypeptide and thus are more likely to maintainShiga toxin effector polypeptide structure and function.

Among certain embodiments of the present invention, the de-immunized,Shiga toxin effector polypeptide comprising, consisting of, orconsisting essentially of amino acids 75 to 251 of SLT-1A (SEQ ID NO:1),StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) wherein at least oneamino acid residue is disrupted in a natively positioned, epitoperegion. Among certain other embodiments are de-immunized, Shiga toxineffector polypeptides which comprise or consist essentially of aminoacids 1 to 241 of SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), and/orSLT-2A (SEQ ID NO:3) wherein at least one amino acid residue isdisrupted in a natively positioned, epitope region. Further embodimentsare de-immunized, Shiga toxin effector polypeptides which comprise,consist of, or consist essentially of amino acids 1 to 251 of SLT-1A(SEQ ID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) whereinat least one amino acid residue is disrupted in a natively positioned,epitope region provided. Further embodiments are Shiga toxin effectorpolypeptides comprising amino acids 1 to 261 of SLT-1A (SEQ ID NO:1),StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) wherein at least oneamino acid residue is disrupted in a natively positioned, epitoperegion. Among certain embodiments of the present invention, thede-immunized, Shiga toxin effector polypeptide comprises, consistsessentially of, or consists of amino acids 75 to 251 of any one of SEQID NOs: 1-6, wherein at least one amino acid residue is disrupted in anatively positioned, epitope region. Among certain other embodiments arede-immunized, Shiga toxin effector polypeptides which comprise, consistessentially of, or consist of amino acids 1 to 241 of SEQ ID NOs: 1-18,wherein at least one amino acid residue is disrupted in a nativelypositioned, epitope region. Further embodiments are de-immunized, Shigatoxin effector polypeptides which comprise, consist essentially of, orconsist of amino acids 1 to 251 of SEQ ID NOs: 1-6, wherein at least oneamino acid residue is disrupted in a natively positioned, epitope regionprovided. Further embodiments are Shiga toxin effector polypeptidescomprising amino acids 1 to 261 of SEQ ID NOs: 1-3, wherein at least oneamino acid residue is disrupted in a natively positioned, epitoperegion. In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide comprises, consists essentially of, or consists of aminoacids 75 to 251 of any one of SEQ ID NOs: 1-6, wherein at least oneamino acid residue is disrupted in a natively positioned, epitoperegion. Among certain further embodiments are de-immunized, Shiga toxineffector polypeptides which comprise, consist essentially of, or consistof amino acids 1 to 241 of SEQ ID NOs: 1-6, wherein at least one aminoacid residue is disrupted in a natively positioned, epitope region.Further embodiments are de-immunized, Shiga toxin effector polypeptideswhich comprise, consist essentially of, or consist of amino acids 1 to251 of SEQ ID NOs: 1-6, wherein at least one amino acid residue isdisrupted in a natively positioned, epitope region provided. Furtherembodiments are Shiga toxin effector polypeptides comprising amino acids1 to 261 of SEQ ID NOs: 1-3, wherein at least one amino acid residue isdisrupted in a natively positioned, epitope region.

There are numerous, diverse, internal amino acid substitutions that canbe used to create de-immunized, Shiga toxin effector polypeptides of theinvention. Of the possible substitute amino acids to use within anepitope region, the following substitute amino acid residues arepredicted to be the most likely to reduce the antigenicity and/orimmunogenicity of an epitope - G, D, E, S, T, R, K, and H. Except forglycine, these amino acid residues may all be classified as polar and/orcharged residues. Of the possible amino acids to substitute with, thefollowing amino acids A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and Kare predicted to be the most likely to reduce antigenicity and/orimmunogenicity while providing the retention of a significant level of aShiga toxin effector function(s), depending on the amino acidsubstituted for. Generally, the substitution should change a polarand/or charged amino acid residue to a non-polar and uncharged residue(see e.g. WO 2015/113007). In addition, it may be beneficial to epitopedisruption to reduce the overall size and/or length of the amino acidresidue’s R-group functional side chain (see e.g. WO 2015/113007).However despite these generalities of substitutions most likely toconfer epitope disruption, because the aim is to preserve significantShiga toxin effector function(s), the substitute amino acid might bemore likely to preserve Shiga toxin effector function(s) if it resemblesthe amino acid substituted for, such as, e.g., a nonpolar and/oruncharged residue of similar size substituted for a polar and/or chargedresidue.

In the Examples below and in WO 2015/113007, many mutations have beenempirically tested for effect(s) on the Shiga toxin effector function ofvarious Shiga toxin effector polypeptides and cell-targeting molecules.Table B summarizes the results described in WO 2015/113007 and WO2016/196344 where an amino acid substitution, alone or in combinationwith one or more other substitutions, did not prevent the exhibition ofa potent level of a Shiga toxin effector function(s). Table B uses theepitope region numbering scheme described in WO 2016/196344.

TABLE B. Amino Acid Substitutions in Shiga Toxin Effector PolypeptidesEpitope Region Disrupted natively positioned amino acid positionsSubstitution B-Cell Epitope Region T-Cell Epitope 1 K1A 1-15 1 K1M 1-151 T4I 1-15 4-33 1 D6R 1-15 4-33 1 S8I 1-15 4-33 1 T9V 1-15 4-33 1 T9I1-15 4-33 1 K11A 1-15 4-33 1 K11H 1-15 4-33 1 T12K 1-15 4-33 2 S33I27-37 4-33 2 S33C 27-37 4-33 3 S43N 39-48 34-78 3 G44L 39-48 34-78 3T45V 39-48 34-78 3 T45I 39-48 34-78 3 S45V 39-48 34-78 3 S45I 39-4834-78 3 G46P 39-48 34-78 3 D47G 39-48 34-78 3 D47M 39-48 34-78 3 N48V39-48 34-78 3 N48F 39-48 34-78 - L49A immunogenic residue 34-78 - F50T34-78 - A51V 34-78 4 D53A 53-66 34-78 4 D53G 53-66 34-78 4 D53N 53-6634-78 4 V54L 53-66 34-78 4 V54I 53-66 34-78 4 R55A 53-66 34-78 4 R55V53-66 34-78 4 R55L 53-66 34-78 4 G56P 53-66 34-78 4 I57M 53-66 34-78 4I57F 53-66 34-78 4 D58A 53-66 34-78 4 D58V 53-66 34-78 4 D58F 53-6634-78 4 P59A 53-66 34-78 4 P59F 53-66 34-78 4 E60I 53-66 34-78 4 E60T53-66 34-78 4 E60R 53-66 34-78 4 E61A 53-66 34-78 4 E61V 53-66 34-78 4E61L 53-66 34-78 4 G62A 53-66 34-78 - R84A 77-103 - V88A 77-103 5 D94A94-115 77-103 5 S96I 94-115 77-103 5 T104N 94-115 5 A105L 94-115 5 T107P94-115 5 L108M 94-115 5 S109V 94-115 5 G110A 94-115 5 D111T 94-115 5S112V 94-115 6 D141A 141-153 128-168 6 G147A 141-153 128-168 - V154A128-168 7 R179A 179-190 160-183 7 T180G 179-190 160-183 7 T181I 179-190160-183 7 D183A 179-190 160-183 7 D183G 179-190 160-183 7 D184A 179-1907 D184F 179-190 7 L185V 179-190 7 S186A 179-190 7 S186F 179-190 7 G187A179-190 7 G187T 179-190 7 R188A 179-190 7 R188L 179-190 7 S189A179-190 - D198A immunogenic residue - R205A immunogenic residue - C242S236-258 8 R248A 243-257 236-258 8 R251A 243-257 236-258

Based on the empirical evidence in WO 2015/113007 and WO 2016/196344,certain amino acid positions in the A Subunits of Shiga toxins arepredicted to tolerate epitope disruptions while still retainingsignificant Shiga toxin effector functions. For example, the followingnatively occurring positions tolerate amino acid substitutions, eitheralone or in combination, while retaining a Shiga toxin effectorfunction(s) such as cytotoxicity - 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2;43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1 or SEQ ID NO:2; 47 ofSEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO:1 or SEQ IDNO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ IDNO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ IDNO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1 or SEQ IDNO:2; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ IDNO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1 or SEQ ID NO:2; 94 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1 orSEQ ID NO:2; 107 of SEQ ID NO:1 or SEQ ID NO:2; 108 of SEQ ID NO:1 orSEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:1 or SEQ ID NO:2;147 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 orSEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ IDNO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ IDNO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2;and 286 of SEQ ID NO:1 or SEQ ID NO:2.

The empirical data in WO 2015/113007 and WO 2016/196344 point towardsother epitope disrupting substitutions and combinations of epitopedisrupting substitutions that can reduce antigenicity and/orimmunogenicity of a Shiga toxin effector polypeptide while retaining theability of the Shiga toxin effector polypeptide to exhibit a significantShiga toxin effector function such as, e.g., new combinations of theaforementioned truncations and positions tolerating substitutions aswell as new substitutions at identical positions or conserved positionsin related Shiga toxin A Subunits.

It is predictable that other amino acid substitutions to amino acidresidues of a conservative functional group of a substitution testedherein may reduce antigenicity and/or immunogenicity while preserving asignificant Shiga toxin effector function. For example, othersubstitutions known to the skilled worker to be similar to any of K1A,K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N,G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T,A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M,D58A, D58V, D58F, P59A, P59F, E60I, E60T, E60R, E61A, E61V, E61L, G62A,R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A,D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G,D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A,R188L, S189A, D198A, R204A, R205A, C242S, R247A, S247I, Y247A, R248A,R250A, R251A, or D264A, G264A, T286A, and/or T286I may disrupt anendogenous epitope while maintaining at least one Shiga toxin effectorfunction. In particular, amino acid substitutions to conservative aminoacid residues similar to K1A, K1M, T4I, S8I, T8V, T9I, S9I, K11A, K11H,S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, N48V, N48F,L49A, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M,D58A, D58V, D58F, P59A, E60I, E60T, E61A, E61V, E61L, G62A, R84A, V88A,D94A, S96I, T104N, T107P, L108M, S109V, T109V, G110A, D111T, S112V,D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184F,L185V, S186A, S186F, G187A, R188A, R188L, S189A, D198A, R204A, R205A,C242S, S247I, Y247A, R247A, R248A, R250A, R251A, D264A, G264A, T286A,and T286I may have the same or similar effects. In certain embodiments,a Shiga toxin effector polypeptide of the invention may comprise similarconservative amino acid substitutions to empirically tested ones, suchas, e.g., K1 to G, V, L, I, F, and H; T4 to A, G, V, L, F, M, and S; S8to A, G, V, L, F, and M; T8 to A, G, V, I, L, F, and M; T9 to A, G, L,F, M, and S; S9 to A, G, L, I, F, and M; K11 to G, V, L, I, F, and M;S33 to A, G, V, L, F, and M; S43 to A, G, V, L, I, F, and M; S45 to A,G, L, F, and M; T45 to A, G, L, F, and M; D47 to A, V, L, I, F, S, andQ; N48 to A, G, L, and M; L49 to G; Y49 to A; D53 to V, L, I, F, S, andQ; R55 to G, I, F, M, Q, S, K, and H; D58 to G, L, I, S, and Q; P59 toG; E60 to A, G, V, L, F, S, Q, N, D, and M; E61 to G, I, F, S, Q, N, D,M, and R; R84 to G, V, L, I, F, M, Q, S, K, and H; V88 to G; I88 to G;D94 to G, V, L, I, F, S, and Q; S96 to A, G, V, L, F, and M; T107 to A,G, V, L, I, F, M, and S; S107 to A, G, V, L, I, F, and M; S109 to A, G,I, L, F, and M; T109 to A, G, I, L, F, M, and S; S112 to A, G, L, I, F,and M; D141 to V, L, I, F, S, and Q; V154 to G; R179 to G, V, L, I, F,M, Q, S, K, and H; T180 to A, V, L, I, F, M, and S; T181 to A, G, V, L,F, M, and S; D183 to V, L, I, F, S, and Q; D184 to G, V, L, I, S, and Q;S186 to G, V, I, L, and M; R188 to G, V, I, F, M, Q, S, K, and H; S189to G, V, I, L, F, and M; D197 to V, L, I, F, S, and Q; D198 to A, V, L,I, F, S, and Q; R204 to G, V, L, I, F, M, Q, S, K, and H; R205 to G, V,L, I, F, M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; Y247 to A,G, V, L, I, F, and M; R247 to A, G, V, L, I, F, M, Q, S, K, and H; R248to G, V, L, I, F, M, Q, S, K, and H; R250 to G, V, L, I, F, M, Q, S, K,and H; R251 to G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L, I,F, S, and Q; and T286 to A, G, V, L, I, F, M, and S.

Similarly, amino acid substitutions which remove charge, polarity,and/or reduce side chain length can disrupt an epitope while maintainingat least one Shiga toxin effector function. In certain embodiments, aShiga toxin effector polypeptide of the invention may comprise one ormore epitopes disrupted by substitutions such that side chain charge isremoved, polarity is removed, and/or side chain length is reduced suchas, e.g., substituting the appropriate amino acid selected from thefollowing group A, G, V, L, I, P, C, M, F, S, D, N, Q, H, or K for theamino acid residue at position 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1 or SEQ ID NO:2; 8of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 12of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ IDNO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ IDNO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1 or SEQ IDNO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO: 1 orSEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ IDNO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ IDNO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ IDNO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ IDNO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2;111 of SEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 141 of SEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2;181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2;189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ IDNO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ IDNO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2;and 286 of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, a Shigatoxin effector polypeptide of the present invention may comprise one ormore of the following amino acid substitutions: K1 to A, G, V, L, I, F,M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S,and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, and S;T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 toA, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, and S; S33 to A,G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to Aand P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;I57 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F,S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 toA, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H;V88 to A and G; I88 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F,M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 toA, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A;D111 to A, G, V, L, I, F, S, and Q; S112 to A, G, V, L, I, F, and M;D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G; R179 toA, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S;T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q;D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G,V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, andH; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S, and Q;D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S,K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V,and S; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M;R247 to A, G, V, L, I, F, M, Q, S, K, and H; R248 to A, G, V, L, I, F,M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 toA, G, V, L, I, F, M, Q, S, K, and H; C262 to A, G, V, and S; D264 to A,G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, andS.

In addition, any amino acid substitution in one epitope region of aShiga toxin effector polypeptide which disrupts an epitope whileretaining significant Shiga toxin effector function is combinable withany other amino acid substitution in the same or a different epitoperegion which disrupts an epitope while retaining significant Shiga toxineffector function to form a de-immunized, Shiga toxin effectorpolypeptide with multiple epitope regions disrupted while stillretaining a significant level of Shiga toxin effector function. Incertain embodiments, a Shiga toxin effector polypeptide of the inventionmay comprise combinations of two or more of the aforementionedsubstitutions and/or the combinations of substitutions described in WO2015/113007 and/or WO 2016/196344.

Based on the empirical evidence in the Examples and in WO 2015/113007and WO 2016/196344, certain amino acid regions in the A Subunits ofShiga toxins are predicted to tolerate epitope disruptions while stillretaining significant Shiga toxin effector functions. For example, theepitope regions natively positioned at 1-15, 39-48, 53-66, 55-66,94-115, 180-190, 179-190, and 243-257 tolerated multiple amino acidsubstitution combinations simultaneously without compromising Shigatoxin enzymatic activity and cytotoxicity.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises, consists essentially of,or consists of an amino acid sequence that is at least 85% (such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to an amino acid sequence selected from any one of SEQ ID NOs:19-21 and 75-89. For example, the de-immunized Shiga toxin effectorpolypeptide of the present invention comprises any of the following setsof substitutions: (i) K1A, S45I, V54I, R55L, I57F, P59F, E60T, E61L,G110A, G147A, C242S, R248A, and R251A; (ii) S45I, V54I, R55L, I57F,P59F, E60T, E61L, G110A, R188A, C242S, R248A, and R251A; or (iii) S45I,V54I, R55L, I57F, P59F, E60T, E61L, G110A, D141A, R188A, C242S, R248A,and R251A.

B. Examples of Furin-Cleavage Resistant, Shiga Toxin EffectorPolypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention may comprise a disrupted, furin cleavage motif and/orfurin cleavage site at the carboxy-terminus of a Shiga toxin A1 fragmentderived region. In certain further embodiments, the Shiga toxin effectorpolypeptide does not comprise any known compensatory structure which mayprovide furin cleavage proximal to the carboxy-terminus of the Shigatoxin A1 fragment derived region. Non-limiting examples of disruptedfurin cleavage motifs and furin cleave sites suitable for use in thepresent invention are described in WO 2015/191764.

Certain furin-cleavage motif disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits provided in the Sequence Listing, noting that naturallyoccurring Shiga toxin A Subunits includes precursor forms containingsignal sequences of about 22 amino acids at their amino-terminals whichare removed to produce mature Shiga toxin A Subunits and arerecognizable to the skilled worker. Further, certain furin-cleavagemotif disruptions comprising mutations are indicated herein by referenceto specific amino acids (e.g. R for an arginine residue) nativelypresent at specific positions within native Shiga toxin A Subunits (e.g.R251 for the arginine residue at position 251 from the amino-terminus)followed by the amino acid with which that residue has been substitutedin the particular mutation under discussion (e.g. R251A represents theamino acid substitution of alanine for arginine at amino acid residue251 from the amino-terminus).

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises a Shiga toxin A1 fragment derived region,wherein the Shiga toxin A1 fragment derived region comprises a disruptedfurin-cleavage motif at the carboxy-terminus of a Shiga toxin A1fragment derived region, and such embodiments are referred to herein as“furin-cleavage resistant” or “protease-cleavage resistant,” Shiga toxineffector polypeptides to describe their property(ies) relative towild-type, Shiga toxin A Subunits and/or wild-type, Shiga toxin A1fragment fusion proteins.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention consists essentially of atruncated Shiga toxin A Subunit having two or more mutations.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention comprises the disruptedfurin-cleavage motif comprising the amino acid residue substitution(relative to a wild-type Shiga toxin polypeptide) of one or both of thearginine residues in the minimal, furin-cleavage site consensus motifwith A, G, or H. In certain embodiments, the protease-cleavageresistant, Shiga toxin effector polypeptide of the present inventioncomprises a disruption which comprises an amino acid substitution withina furin-cleavage motif region, wherein the substitution occurs at thenatively positioned amino acid selected from the group consisting of:247 of SEQ ID NO:3, 248 of SEQ ID NO: 1 or SEQ ID NO:2, 250 of SEQ IDNO:3, 251 of SEQ ID NO: 1 or SEQ ID NO:2, or the equivalent position ina conserved Shiga toxin effector polypeptide and/or non-native Shigatoxin effector polypeptide sequence, such as, e.g., position 247 of SEQID NOs: 7-18, 248 of SEQ ID NOs: 4-6, 250 of SEQ ID NOs: 7-18, or 251 ofSEQ ID NOs: 4-6. In certain further embodiments, the substitution is toany non-conservative amino acid and the substitution occurs at thenatively positioned amino acid residue position. In certain furtherembodiments, the mutation comprises an amino acid substitution selectedfrom the group consisting of: R247A, R248A, R250A R251A, or theequivalent position in a conserved Shiga toxin effector polypeptideand/or non-native Shiga toxin effector polypeptide sequence.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention comprises the disruptedfurin-cleavage motif comprising the mutation which is a deletion. Incertain further embodiments, the disrupted furin-cleavage motifcomprises a mutation which is a deletion of the region nativelypositioned at 247-252 in StxA (SEQ ID NO:2), SLT-1A (SEQ ID NO:1), andother Shiga toxin 1 A Subunit variants (e.g. SEQ ID NOs: 4-6), or theregion natively positioned at 246-251 in SLT-2A (SEQ ID NO:3) andShiga-like toxin 2 A Subunit variants (e.g. SEQ ID NOs: 7-18); adeletion of the region natively positioned at 244-246 in StxA (SEQ IDNO:2), SLT-1A (SEQ ID NO:1), and other Shiga toxin 1 A Subunit variants(e.g. SEQ ID NOs: 4-6), or the region natively positioned at 243-245 inSLT-2A (SEQ ID NO:3) and Shiga-like toxin 2 A Subunit variants (e.g. SEQID NOs: 7-18); or a deletion of the region natively positioned at253-259 in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:3), or the regionnatively positioned at 252-258 in SLT-2A (SEQ ID NO:3).

In certain embodiments of the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention comprises a Shiga toxin A1fragment region comprising a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment region that is disruptedby a carboxy-terminal truncation as compared to the carboxy-terminus ofa wild-type Shiga toxin A Subunit, and wherein the truncation results inthe deletion of one or more amino acid residues within thefurin-cleavage motif as compared to the wild-type Shiga toxin A Subunit.In certain further embodiments, the disrupted furin-cleavage motifcomprises the carboxy-terminal truncation which deletes one or moreamino acid residues within the minimal cleavage site Y/R-x-x-R, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides,truncations ending at the natively amino acid residue position 250, 249,248, 247, 246, 245, 244, 243, 242, 241, 240, or less; and for SLT-2Aderived Shiga toxin effector polypeptides, truncations ending at thenatively amino acid residue position 249, 248, 247, 246, 245, 244, 243,242, 241, or less. Certain further embodiments comprise the disruptedfurin-cleavage motif comprising a combination of any of theaforementioned mutations, where possible.

In certain embodiments, the disrupted furin-cleavage motif comprises themutation(s) that is a partial, carboxy-terminal truncation of thefurin-cleavage motif; however, certain molecules of the presentinvention do not comprise the disrupted furin-cleavage motif which is acomplete, carboxy-terminal truncation of the entire 20 amino acidresidue, furin-cleavage motif. For example, certain, Shiga toxineffector polypeptides of the present invention comprise the disruptedfurin-cleavage motif comprising a partial, carboxy-terminal truncationof the Shiga toxin A1 fragment region up to native position 240 in StxA(SEQ ID NO:2), SLT-1A (SEQ ID NO:1), or another Shiga toxin 1 A Subunitvariant (e.g. SEQ ID NOs: 4-6) but not a carboxy-terminal truncation atposition 239 or less. Similarly, certain, certain, Shiga toxin effectorpolypeptides of the present invention comprise the disruptedfurin-cleavage motif comprising a partial, carboxy-terminal truncationof the Shiga toxin A1 fragment region up to native position 239 inSLT-2A (SEQ ID NO:3) or a Shiga-like toxin 2 A Subunit variant (e.g. SEQID NOs: 7-18) but not a carboxy-terminal truncation at position 238 orless. In the largest carboxy-terminal truncation of the furin-cleavageresistant, Shiga toxin effector polypeptide of the present invention,mutations comprising the disrupted furin-cleavage motif, positions P14and P13 of the furin-cleavage motif are still present.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue substitution within the furin-cleavage motifand a carboxy-terminal truncation as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises both an amino acid residue substitutionwithin the minimal furin-cleavage site R/Y-x-x-R and a carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides (andShiga toxin 1 A Subunit variants), truncations ending at the nativelyamino acid residue position 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, or greater and comprising the nativelypositioned amino acid residue R248 and/or R251 substituted with anynon-positively charged, amino acid residue where appropriate; and forSLT-2A derived Shiga toxin effector polypeptides (and Shiga-like toxin 2A Subunit variants), truncations ending at the natively amino acidresidue position 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, or greater and comprising the nativelypositioned amino acid residue R/Y247 and/or R250 substituted with anynon-positively charged, amino acid residue where appropriate. In certainembodiments, the truncated Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif also comprises the furin-cleavage motif,amino acid residues at positions P9, P8, and/or P7 in order to maintainoptimal cytotoxicity.

In certain embodiments, the disrupted furin-cleavage motif comprises amutation(s) which is one or more internal, amino acid residue deletions,as compared to a wild-type, Shiga toxin A Subunit. In certain furtherembodiments, the disrupted furin-cleavage motif comprises a mutation(s)which has one or more amino acid residue deletions within the minimalfurin-cleavage site R/Y-x-x-R. For example, StxA and SLT-1A derivedShiga toxin effector polypeptides (and other Shiga toxin 1 A Subunitvariants) comprising internal deletions of the natively positioned aminoacid residues R248 and/or R251, which may be combined with deletions ofsurrounding residues such as, e.g., 249, 250, 247, 252, etc.; and SLT-2Aderived Shiga toxin effector polypeptides (and Shiga-like toxin 2 ASubunit variants) comprising internal deletions of the nativelypositioned amino acid residues R/Y247 and/or R250, which may be combinedwith deletions of surrounding residues such as, e.g., 248, 249, 246,251, etc. In certain further embodiments, the disrupted furin-cleavagemotif comprises a mutation which is a deletion of four, consecutive,amino acid residues which deletes the minimal furin-cleavage siteR/Y-x-x-R, such as, e.g., StxA and SLT-1A derived Shiga toxin effectorpolypeptides (and other Shiga toxin 1 A Subunit variants) lackingR248-R251 and SLT-2A derived Shiga toxin effector polypeptides (andShiga-like toxin 2 A Subunit variants) lacking R/Y247-R250. In certainfurther embodiments, the disrupted furin-cleavage motif comprises amutation(s) having one or more amino acid residue deletions in the aminoacid residues flanking the core furin-cleavage motif, such as, e.g., adeletion of 244-247 and/or 252-255 in SLT-1A, StxA, or another Shigatoxin 1 A Subunit variant. In certain further embodiments, the disruptedfurin-cleavage motif comprises a mutation which is an internal deletionof the entire surface-exposed, protease-cleavage sensitive loop ascompared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxAand SLT-1A derived Shiga toxin effector polypeptides (and other Shigatoxin 1 A Subunit variants), a deletion of natively positioned aminoacid residues 241-262; and for SLT-2A derived Shiga toxin effectorpolypeptides, a deletion of natively positioned amino acid residues240-261.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth a mutation which is an internal, amino acid residue deletion withinthe furin-cleavage motif and a mutation which is carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit. In certainfurther embodiments, the disrupted furin-cleavage motif comprises both amutation which is an amino acid residue deletion within the minimalfurin-cleavage site R/Y-x-x-R and a mutation which is a carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit. Forexample, protease-cleavage resistant, Shiga toxin effector polypeptidesmay comprise a disrupted furin-cleavage motif comprising mutation(s)which are deletions of the natively positioned amino acid residues248-249 and/or 250-251 in a truncated StxA or SLT-1A polypeptide (oranother Shiga toxin 1 A Subunit variant) which still has amino acidresidue 247 and/or 252, or the amino acid residues 247-248 and/or249-250 in a truncated SLT-2A (or a Shiga-like toxin 2 A Subunitvariant) which still has amino acid residue 246 and/or 251. In certainfurther embodiments, the disrupted furin-cleavage motif comprises amutation having a deletion of four, consecutive, amino acid residueswhich deletes the minimal furin-cleavage site R/Y-x-x-R and acarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit, such as, e.g., for StxA and SLT-1A (and other Shiga toxin 1 ASubunit variants) derived Shiga toxin effector polypeptides, truncationsending at the natively amino acid residue position 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, or greater and lackingR248-R251; and for SLT-2A derived Shiga toxin effector polypeptides (andShiga toxin 2 A Subunit variants), truncations ending at the nativelyamino acid residue position 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, or greater and lacking R/Y247-R250.

C. Examples of Shiga Toxin Effector Polypeptides Having an EmbeddedEpitope

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention may comprise one or more embedded or inserted,heterologous, T-cell epitopes for purposes of de-immunization and/ordelivery to a MHC class I presentation pathway of a target cell. Forcertain embodiments and/or certain Shiga toxin effector polypeptidesub-regions, embedding or partial embedding a T-cell epitope may bepreferred over inserting a T-cell epitope because, e.g., embedding-typemodifications are more likely to be successful in diverse sub-regions ofa Shiga toxin effector polypeptide whereas successful insertions may bemore limited to a smaller subset of Shiga toxin effector polypeptidesub-regions. The term “successful” is used here to mean the modificationto the Shiga toxin effector polypeptide (e.g. introduction of aheterologous, T-cell epitope) results in a modified Shiga toxin effectorpolypeptide which retains one or more Shiga toxin effector functions atthe requisite level of activity either alone or as a component of acell-targeting molecule.

Any of the Shiga toxin effector polypeptide sub-regions described in WO2015/113007 may be suitable for certain embodiments of the presentinvention, and any of the Shiga toxin effector polypeptides described inWO 2015/113007 may be modified into a Shiga toxin effector polypeptideof the present invention, e.g., by the addition of one or more newepitope region disruptions for de-immunization (such one as describedherein) and/or a furin-cleavage motif disruption (such as one describedherein).

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention consists essentially of a truncated Shiga toxin ASubunit comprising an embedded or inserted, heterologous, T-cell epitopeand one or more other mutations. In certain embodiments, the Shiga toxineffector polypeptide of the present invention comprises an embedded orinserted, heterologous, T-cell epitope and is smaller than afull-length, Shiga toxin A Subunit, such as, e.g., derived from thepolypeptide represented by amino acids 77 to 239 of SLT-1A (SEQ ID NO:1)or StxA (SEQ ID NO:2) or the equivalent in other A Subunits of membersof the Shiga toxin family (e.g. amino acids 77 to 238 of SLT-2A (SEQ IDNO:3)). For example, the Shiga toxin effector polypeptide of the presentinvention comprising an embedded or inserted, heterologous, T-cellepitope may comprise, consist essentially of, or consist of an aminoacid sequence that is at least 85% (such as at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acidsequence selected from any one of SEQ ID NOs: 19-21 and 75-89. Forexample, the Shiga toxin effector polypeptide of the present inventioncomprising an embedded, heterologous epitope comprises: V54I, R55L,I57F, P59F, E60T, and E61L.

D. Examples of Combination Shiga Toxin Effector Polypeptides

A combination Shiga toxin effector polypeptide of the present inventioncomprises two or more sub-regions (i.e. non-overlapping sub-regions)wherein each sub-region comprises at least one of the following: (1) adisruption in an endogenous epitope or epitope region; (2) an embedded,heterologous, T-cell epitope-peptide; (3) an inserted, heterologous,T-cell epitope-peptide; and (4) a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region. In certainfurther embodiments, the combination Shiga toxin effector polypeptidecomprises a carboxy-terminal truncation relative to a wild-type Shigatoxin A Subunit. In certain further embodiments, the carboxy-terminaltruncation results in the removal of one or more endogenous, B-celland/or CD4+ T-cell epitope regions present in an untruncated, wild-typeShiga toxin A Subunit.

Certain embodiments of the combination Shiga toxin effector polypeptidesof the present invention comprise both (1) a disruption in an endogenousepitope or epitope region and (2) a disrupted furin-cleavage motif atthe carboxy-terminus of an A1 fragment derived region. It is predictedthat any of the individual, de-immunized, Shiga toxin effectorsub-regions described in WO 2015/113007 and WO 2016/196344 (see e.g.Table B, supra) may generally be combined with any Shiga toxin effectorsub-region comprising a disrupted furin-cleavage motif described herein,described in WO 2015/191764, and/or known in the art in order to createa Shiga toxin effector polypeptide of the present invention.

In certain embodiments of the present invention, the Shiga toxineffector polypeptide consists essentially of the polypeptide shown inSEQ ID NO:37 which further comprises a disruption of at least one,endogenous, B-cell and/or T-cell epitope region which does not overlapwith an embedded or inserted, heterologous, CD8+ T-cell epitope; whereinthe disruption comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin. In certain further embodiments thesubstitution is selected from the group consisting of: K1 to A, G, V, L,I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F,S, Q and R; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, andM; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to A,G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S,M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; Y49 to A,G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; D53 to A, G, V, L,I, F, S, and Q; V54 to A, G, I, and L; R55 to A, G, V, L, I, F, M, Q, S,K, and H; G56 to A and P; I57 to A, G, V, and M; L57 to A, V, C, G, M,and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A,G, V, L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L, I, F, S, Q,N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, and H;V88 to A and G; I88 to A, V, C, and G; D94 to A, G, V, L, I, F, S, andQ; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M; and N;A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G, V, L, I, F,M, and P; L108 to A, V, C, and G; S109 to A, G, V, I, L, F, and M; T109to A, G, V, I, L, F, M, and S; G110 to A; S112 to A, G, V, L, I, F, andM; D111 to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V, L, I, F, andM; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G. R179to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, andS; T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, andQ; D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, V and C; S186 to A,G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K,and H; S189 to A, G, V, I, L, F, and M; D198 to A, G, V, L, I, F, S, andQ; R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V, L, I,F, M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V,L, I, F, and M; R247 to A, G, V, L, I, F, M, Q, S, K, and H; R248 to A,G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K,and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L,I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S. Incertain further embodiments, there are multiple disruptions of multiple,endogenous B-cell and/or CD8+ T-cell epitope regions wherein eachdisruption involves at least one amino acid residue substitutionselected from the group consisting of: K1 to A, G, V, L, I, F, M and H;T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, and S; S9 to A,G, V, L, I, F, and M; K11 to A, G, V, L, I, F, M and H; T12 to A, G, V,I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L,I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A or L; S45 to A,G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to A and P;D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, M and F; L49 toA, V, C, and G; Y49 to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I,and T; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A, G, I, andL; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P; 157 to A,G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L, I, F, S,and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M, T,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; R84 toA, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; I88 to A, V, C, andG; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, L, I, F, M; and N; A105 to L; T107 to A, G, V, L, I, F,M, and P; S107 to A, G, V, L, I, F, M, and P; L108 to A, V, C, and G;S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S;G110 to A; S112 to A, G, V, L, I, F, and M; D111 to A, G, V, L, I, F, S,Q, and T; S112 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F, S,and Q; G147 to A; V154 to A and G. R179 to A, G, V, L, I, F, M, Q, S, K,and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F, M,and S; D183 to A, G, V, L, I, F, S, and Q; D184 to A, G, V, L, I, F, S,and Q; L185 to A, G, V and C; S186 to A, G, V, I, L, F, and M; G187 toA; R188 to A, G, V, L, I, F, M, Q, S, K, and H; S189 to A, G, V, I, L,F, and M; D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F,M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; S247 toA, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R247 to A, G,V, L, I, F, M, Q, S, K, and H; R248 to A, G, V, L, I, F, M, Q, S, K, andH; R250 to A, G, V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I,F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; G264 to A; andT286 to A, G, V, L, I, F, M, and S.

Certain embodiments of the Shiga toxin effector polypeptides of thepresent invention comprise both (1) an embedded or inserted,heterologous, T-cell epitope-peptide and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of an A1 fragment derived region. Any ofthe Shiga toxin effector polypeptide sub-regions comprising an embeddedor inserted, heterologous, T-cell epitope described in the Examplesbelow or in WO 2015/113007 may generally be combined with anyprotease-cleavage resistant, Shiga toxin effector polypeptide sub-region(e.g., modified, Shiga toxin A Subunit sub-regions described herein,described in WO 2015/191764, and/or known in the art) in order to createa combination, Shiga toxin effector polypeptide which, as a component ofa cell-targeting molecule, is both protease-cleavage resistant andcapable of delivering a heterologous, T-cell epitope to the MHC class Ipresentation pathway of a target cell. Non-limiting examples of thistype of combination Shiga toxin effector polypeptide are shown in SEQ IDNOs: 19-21 and 75-89.

In certain embodiments of the present invention, the Shiga toxineffector polypeptide comprises an embedded or inserted, heterologous,T-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region. Forexample in certain embodiments, the Shiga toxin effector polypeptide ofthe present invention is derived from amino acids 75 to 251 of SEQ IDNO:1, 1 to 241 of SEQ ID NO:1, 1 to 251 of SEQ ID NO:1, or amino acids 1to 261 of SEQ ID NO:1, wherein the Shiga toxin effector polypeptidecomprises at least one embedded or inserted, heterologous T-cell epitopeand a disrupted furin-cleavage motif at the carboxy-terminus of a Shigatoxin A1 fragment derived region. Similarly in other embodiments, theShiga toxin effector polypeptide of the present invention is derivedfrom amino acids 75 to 251 of SEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to251 of SEQ ID NO:2, or amino acids 1 to 261 of SEQ ID NO:2, wherein theShiga toxin effector polypeptide comprises at least one embedded orinserted, heterologous T-cell epitope and a disrupted furin-cleavagemotif at the carboxy-terminus of a Shiga toxin A1 fragment derivedregion. Additionally, the Shiga toxin effector polypeptide may bederived from amino acids 75 to 251 of SEQ ID NO:3, 1 to 241 of SEQ IDNO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to 261 of SEQ ID NO:3,wherein the Shiga toxin effector polypeptide comprises at least oneembedded or inserted, heterologous T-cell epitope and a disruptedfurin-cleavage motif at the carboxy-terminus of a Shiga toxin A1fragment derived region. In certain embodiments, the Shiga toxineffector polypeptide comprises, consists essentially of, or consists of(i) amino acids 75 to 251 of any one of SEQ ID NOs: 1-18; (ii) aminoacids 1 to 241 of any one of SEQ ID NOs: 1-18; (iii) amino acids 1 to251 of any one of SEQ ID NOs: 1-18; and (iv) amino acids 1 to 261 of anyone of SEQ ID NOs: 1-18, wherein the Shiga toxin effector polypeptidecomprises at least one embedded or inserted, heterologous T-cell epitopeand a disrupted furin-cleavage motif at the carboxy-terminus of a Shigatoxin A1 fragment derived region. In certain embodiments, the Shigatoxin effector polypeptide comprises, consists essentially of, orconsists of: (i) amino acids 75 to 251 of SEQ ID NOs: 1-6, (ii) 1 to 241of SEQ ID NOs: 1-18, (iii) 1 to 251 of SEQ ID NOs: 1-6, or (iv) aminoacids 1 to 261 of SEQ ID NOs: 1-3, wherein the Shiga toxin effectorpolypeptide comprises at least one embedded or inserted, heterologous,CD8+ T-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region. In certainembodiments, the Shiga toxin effector polypeptide comprises, consistsessentially of, or consists of: (i) amino acids 75 to 251 of any one ofSEQ ID NOs: 1-6; (ii) amino acids 1 to 241 of any one of SEQ ID NOs:1-18 and 75-89; (iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6and 75-89; or (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-3;wherein the Shiga toxin effector polypeptide comprises at least oneembedded or inserted, heterologous T-cell epitope and a disruptedfurin-cleavage motif at the carboxy-terminus of a Shiga toxin A1fragment derived region. In certain embodiments, the Shiga toxineffector polypeptide comprises, consists essentially of, or consists of:(i) amino acids 75 to 251 of any one of SEQ ID NOs: 1-6; (ii) aminoacids 1 to 241 of any one of SEQ ID NOs: 1-18 and 75-89; (iii) aminoacids 1 to 251 of any one of SEQ ID NOs: 1-6 and 75-89; or (iv) aminoacids 1 to 261 of any one of SEQ ID NOs: 1-3; wherein the Shiga toxineffector polypeptide comprises at least one embedded or inserted,heterologous T-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region.

Certain embodiments of the combination Shiga toxin effector polypeptidesof the present invention comprise both (1) a disruption in an endogenousepitope or epitope region and (2) an embedded, heterologous, T-cellepitope-peptide. However, the Shiga toxin effector sub-regionscomprising inserted or embedded, heterologous, T-cell epitopes describedherein or in WO 2015/191764 are generally not combinable with everyde-immunized, Shiga toxin effector sub-regions described herein, exceptwhere empirically shown to be successfully combined such that theresulting combination molecule retained a sufficient level of a Shigatoxin effector function(s). The disclosure herein shows how suchembodiments may be made and tested to empirically demonstrate success.

The term “successful” is used here to mean two or more amino acidresidue substitutions in a Shiga toxin effector polypeptide results in afunctional feature, such as, e.g., de-immunization, reducedfurin-cleavage, and/or ability to deliver an embedded or insertedepitope, while the modified Shiga toxin effector polypeptide retains oneor more Shiga toxin effector functions. The approaches and assaysdescribed herein show how to design, make and empirically testembodiments of the present invention, which represent combination, Shigatoxin effector polypeptides and cell-targeting molecules comprising thesame.

For example, in certain embodiments of the present invention, the Shigatoxin effector polypeptides is derived from amino acids 75 to 251 of SEQID NO: 1, 1 to 241 of SEQ ID NO: 1, 1 to 251 of SEQ ID NO: 1, or aminoacids 1 to 261 of SEQ ID NO:1, wherein the Shiga toxin effectorpolypeptide comprises at least one embedded or inserted, heterologousT-cell epitope and at least one amino acid is disrupted in anendogenous, B-cell and/or CD4+ T-cell epitope region and wherein thedisrupted amino acid does not overlap with the embedded or insertedepitope. Similarly in other embodiments, the Shiga toxin effectorpolypeptide of the present invention is derived from amino acids 75 to251 of SEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, oramino acids 1 to 261 of SEQ ID NO:2, wherein the Shiga toxin effectorpolypeptide comprises at least one embedded or inserted, heterologousT-cell epitope and at least one amino acid is disrupted in anendogenous, B-cell and/or CD4+ T-cell epitope region and wherein thedisrupted amino acid does not overlap with the embedded or insertedepitope. Additionally, the Shiga toxin effector polypeptide may bederived from amino acids 75 to 251 of SEQ ID NO:3, 1 to 241 of SEQ IDNO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to 261 of SEQ ID NO:3,wherein the Shiga toxin effector polypeptide comprises at least oneembedded or inserted, heterologous T-cell epitope and at least one aminoacid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitoperegion and wherein the disrupted amino acid does not overlap with theembedded or inserted epitope. In certain embodiments, the Shiga toxineffector polypeptide is derived from the polypeptide that comprises,consists essentially of, or consists of (i) amino acids 75 to 251 of anyone of SEQ ID NOs: 1-6; (ii) amino acids 1 to 241 of any one of SEQ IDNOs: 1-18; (iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6; and(iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-3, wherein theShiga toxin effector polypeptide comprises at least one embedded orinserted, heterologous T-cell epitope and at least one amino acid isdisrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region andwherein the disrupted amino acid does not overlap with the embedded orinserted epitope. In certain embodiments, the Shiga toxin effectorpolypeptide is derived from the polypeptide that comprises, consistsessentially of, or consists of: (i) amino acids 75 to 251 of SEQ ID NOs:1-6, (ii) 1 to 241 of SEQ ID NOs: 1-18, (iii) 1 to 251 of SEQ ID NOs:1-6, or (iv) amino acids 1 to 261 of SEQ ID NOs: 1-3, wherein the Shigatoxin effector polypeptide comprises at least one embedded or inserted,heterologous T-cell epitope and at least one amino acid is disrupted inan endogenous, B-cell and/or CD4+ T-cell epitope region and wherein thedisrupted amino acid does not overlap with the embedded or insertedepitope and wherein the embedded or inserted, heterologous T-cellepitope disrupts an additional endogenous, B-cell and/or CD4+ T-cellepitope region.

The combination, Shiga toxin effector polypeptides of the presentinvention combine the features of their respective sub-regions, such as,e.g., a furin-cleavage motif disruption, individual epitope disruptions,and/or a heterologous T-cell epitope cargo, and these combinationssometimes result in Shiga toxin effector polypeptides with synergisticreductions in immunogenicity as compared to the sum of their partiallyde-immunized sub-regions. In particular, the exemplary, Shiga toxineffector polypeptides shown in SEQ ID NOs: 19-21 and 75-89 aresynergistically de-immunized due to the combination of two or moresub-regions, one of which comprises an embedded, heterologous, T-cellepitope and another of which comprises an endogenous epitope disruptedby one or more amino acid residue substitutions.

In certain embodiments, the combination, de-immunized, protease-cleavageresistant, Shiga toxin effector polypeptides comprising embedded, T-cellepitopes of the present invention comprises one or more substitutionsselected from the group of substitutions at native positions in a Shigatoxin A Subunit consisting of K1R and K11R.

In certain embodiments, the combination, de-immunized, protease-cleavageresistant, Shiga toxin effector polypeptides comprising embedded, T-cellepitopes of the present invention comprise, consist of, or consistessentially of one of the polypeptides represented by the polypeptidesequence shown in any one of SEQ ID NOs: 19-21 and 75-89, represented byamino acids 2 to 252 of SEQ ID NO:35, or represented by amino acids 1 to251 of SEQ ID NO: 107.

De-immunized, Shiga toxin effector polypeptides of the present inventionwhich exhibit no cytotoxicity or reduced cytotoxicity at certainconcentrations, e.g. Shiga toxin effector polypeptides comprising R179A,may still be useful as de-immunized, Shiga toxin effector polypeptidesfor delivering exogenous materials into cells. Similarly, CD8+ T-cellhyper-immunized, Shiga toxin effector polypeptides of the presentinvention which exhibit no cytotoxicity or reduced cytotoxicity atcertain concentrations, e.g. a Shiga toxin effector polypeptidecomprising an epitope embedded into its catalytic domain (see e.g. WO2015/113005, Example 1-F), may still be useful for delivering a T-cellepitope(s) to a desired subcellular compartment of a cell in which theShiga toxin effector polypeptide is present or as a component of acell-targeting molecule for delivery of a T-cell epitope(s) into atarget cell.

E. Examples of Cell-Targeting Molecules of the Present Invention

The Shiga toxin effector polypeptides described herein may be used ascomponents of cell-targeting molecules that target various HER2 targetbiomolecules and epitopes with the aforementioned. The followingexamples describe in more detail certain structures of exemplarycell-target molecules of the present invention which target cellsphysically coupled to HER2 at a cellular surface, e.g. cells whichexpress HER2. The cell-targeting molecule of the present invention maybe a HER2-targeting molecule comprising (i) an immunoglobulin bindingregion capable of specifically binding an extracellular part ofHER2/neu/ErbB2, and comprising one or more of: an antibody variablefragment, a single-domain antibody fragment, a single-chain variablefragment, a Fd fragment, an antigen-binding fragment, an autonomous VHdomain, a V_(H)H fragment derived from a camelid antibody, a heavy-chainantibody domain derived from a cartilaginous fish antibody, a VNARfragment, and an immunoglobulin new antigen receptor; and (ii) a Shigatoxin A Subunit effector polypeptide comprising a Shiga toxin A1fragment region, wherein the Shiga toxin A subunit effector polypeptidecomprises: (a) an embedded or inserted, heterologous, CD8+ T-cellepitope which disrupts an endogenous, B-cell and/or CD4+ T-cell epitoperegion within the Shiga toxin A1 fragment region; and (b) a disruptionof a plurality of endogenous, B-cell and/or CD4+ T-cell epitope regionswithin the Shiga toxin A1 fragment region which do not overlap with theembedded or inserted, heterologous, CD8+ T-cell epitope; wherein theShiga toxin A1 fragment region comprises a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment region;wherein the Shiga toxin A subunit effector polypeptide comprises acarboxy-terminal truncation as compared to the carboxy-terminus of awild-type Shiga toxin A Subunit; wherein the carboxy-terminal truncationresults in the removal of one or more endogenous, B-cell and/or CD4+T-cell epitope regions described herein; wherein the Shiga toxin Asubunit effector polypeptide is capable of exhibiting a Shiga toxineffector function (e.g. catalytic activity); wherein the HER2-targetingmolecule has reduced B-cell antigenicity or immunogenicity and/orreduced CD4+ T-cell antigenicity or immunogenicity; and wherein thebinding region and the Shiga toxin effector polypeptide are fusedforming a continuous polypeptide such that the binding region isassociated with the carboxy-terminus of the Shiga toxin A subuniteffector polypeptide.

Other Structural Variations

It is within the scope of the present invention to use fragments,variants, and/or derivatives of the cell-targeting molecules of thepresent invention which contain a functional binding site to anyextracellular part of a target biomolecule, and even more preferablycapable of binding a target biomolecule with high affinity (e.g. asshown by K_(D)). For example, any binding region which binds anextracellular part of a target biomolecule with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter, preferably less than 200 nM, maybe substituted for use in making cell-targeting molecules of theinvention and methods of the invention.

The skilled worker will recognize that variations may be made to theShiga toxin effector polypeptides and cell-targeting molecules of thepresent invention, and polynucleotides encoding any of the former,without diminishing their biological activities, e.g., by maintainingthe overall structure and function of the Shiga toxin effectorpolypeptide, such as in conjunction with one or more 1) endogenousepitope disruptions which reduce antigenic and/or immunogenic potential,2) furin-cleavage motif disruptions which reduce proteolytic cleavage,and/or 3) embedded or inserted epitopes which reduce antigenic and/orimmunogenic potential or are capable of being delivered to a MHC Imolecule for presentation on a cell surface. For example, somemodifications may facilitate expression, facilitate purification,improve pharmacokinetic properties, and/or improve immunogenicity. Suchmodifications are well known to the skilled worker and include, forexample, a methionine added at the amino-terminus to provide aninitiation site, additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons, andbiochemical affinity tags fused to either terminus to provide forconvenient detection and/or purification. A common modification toimprove the immunogenicity of a polypeptide produced using anon-chordate system (e.g. a prokaryotic cell) is to remove, after theproduction of the polypeptide, the starting methionine residue, whichmay be formylated during production, such as, e.g., in a bacterial hostsystem, because, e.g., the presence of N-formylmethionine (fMet) mightinduce undesirable immune responses in chordates.

Also contemplated herein is the inclusion of additional amino acidresidues at the amino and/or carboxy termini of a Shiga toxin effectorpolypeptide of the present invention, a cell-targeting molecule of thepresent invention, or a proteinaceous component of a cell-targetingmolecules of the present invention, such as sequences for epitope tagsor other moieties. The additional amino acid residues may be used forvarious purposes including, e.g., facilitating cloning, facilitatingexpression, post-translational modification, facilitating synthesis,purification, facilitating detection, and administration. Non-limitingexamples of epitope tags and moieties are chitin binding proteindomains, enteropeptidase cleavage sites, Factor Xa cleavage sites, FIAsHtags, FLAG tags, green fluorescent proteins (GFP),glutathione-S-transferase moieties, HA tags, maltose binding proteindomains, myc tags, polyhistidine tags, ReAsH tags, strep-tags, strep-tagII, TEV protease sites, thioredoxin domains, thrombin cleavage site, andV5 epitope tags.

In certain of the above embodiments, the polypeptide sequence of theShiga toxin effector polypeptides and/or cell-targeting molecules of thepresent invention are varied by one or more conservative amino acidsubstitutions introduced into the polypeptide region(s) as long as allrequired structural features are still present and the Shiga toxineffector polypeptide is capable of exhibiting any required function(s),either alone or as a component of a cell-targeting molecule. As usedherein, the term “conservative substitution” denotes that one or moreamino acids are replaced by another, biologically similar amino acidresidue. Examples include substitution of amino acid residues withsimilar characteristics, e.g. small amino acids, acidic amino acids,polar amino acids, basic amino acids, hydrophobic amino acids andaromatic amino acids (see, for example, Table C). An example of aconservative substitution with a residue normally not found inendogenous, mammalian peptides and proteins is the conservativesubstitution of an arginine or lysine residue with, for example,ornithine, canavanine, aminoethylcysteine, or another basic amino acid.For further information concerning phenotypically silent substitutionsin peptides and proteins see, e.g., Bowie J et al., Science 247: 1306-10(1990).

TABLE C. Examples of Conservative Amino Acid Substitutions I II III IV VVI VII VIII IX X XI XII XIII XIV A D H C F N A C F A C A A D G E K I W QG M H C D C C E P Q R L Y S I P W F E D D G S N M T L Y G H G E K T V VH K N G P I N P H Q L Q S K R M R T N S R S V Q T T T R V S W P Y T

In the conservative substitution scheme in Table C, exemplaryconservative substitutions of amino acids are grouped by physicochemicalproperties - I: neutral, hydrophilic; II: acids and amides; III: basic;IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilicuncharged, VII aliphatic uncharged, VIII non-polar uncharged, IXcycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIIIturn-permitting, and XIV flexible. For example, conservative amino acidsubstitutions include the following: 1) S may be substituted for C; 2) Mor L may be substituted for F; 3) Y may be substituted for M; 4) Q or Emay be substituted for K; 5) N or Q may be substituted for H; and 6) Hmay be substituted for N.

Additional conservative amino acid substitutions include thefollowing: 1) S may be substituted for C; 2) M or L may be substitutedfor F; 3) Y may be substituted for M; 4) Q or E may be substituted forK; 5) N or Q may be substituted for H; and 6) H may be substituted forN.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention may comprisefunctional fragments or variants of a polypeptide region of the presentinvention described herein that have, at most, 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid substitutions compared to a polypeptidesequence recited herein (and which retain at least 85%, 90%, 95%, 96%,97%, 98%, 99% or more amino acid sequence identity to the polypeptidesequences recited herein), as long as it (1) comprises at least oneembedded or inserted, heterologous T-cell epitope and at least one aminoacid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitoperegion, wherein the disrupted amino acid does not overlap with theembedded or inserted epitope; (2) comprises at least one embedded orinserted, heterologous T-cell epitope and a disrupted furin-cleavagemotif at the carboxy-terminus of a Shiga toxin A1 fragment derivedregion; (3) comprises a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region andcomprises at least one amino acid is disrupted in an endogenous, B-celland/or CD4+ T-cell epitope region, wherein the disrupted amino acid doesnot overlap with the disrupted furin-cleavage motif; or (4) comprises atleast one embedded or inserted, heterologous T-cell epitope, at leastone amino acid is disrupted in an endogenous, B-cell and/or CD4+ T-cellepitope region, wherein the disrupted amino acid does not overlap withthe embedded or inserted epitope, and a disrupted furin-cleavage motifat the carboxy-terminus of a Shiga toxin A1 fragment derived region.Variants of the Shiga toxin effector polypeptides and cell-targetingmolecules of the invention are within the scope of the present inventionas a result of changing a polypeptide described herein by altering oneor more amino acid residues or deleting or inserting one or more aminoacid residues, such as within the binding region or Shiga toxin effectorpolypeptide region, in order to achieve desired properties, such aschanged cytotoxicity, changed cytostatic effects, changedimmunogenicity, and/or changed serum half-life. The Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention mayfurther be with or without a signal sequence.

Accordingly, in certain embodiments, the cell-targeting molecule of thepresent invention comprises, consists essentially of, or consists of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acidsequence shown in any one of SEQ ID NOs: 22-36 and 97-108. In certainembodiments, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceshown in any one of SEQ ID NOs: 25-31, 34-36, 97-104, and 106-108. Incertain embodiments, the cell-targeting molecule of the presentinvention comprises, consists essentially of, or consists of an aminoacid sequence that is at least 85% (such as at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acidsequence shown in any one of SEQ ID NOs: 29, 31, 34, 35, 36, 102, 104,and 106-108. In certain embodiments, the cell-targeting molecule of thepresent invention comprises, consists essentially of, or consists of anamino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence shown in any one of SEQ ID NOs: 29 or 102. In certainembodiments, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceshown in any one of SEQ ID NOs: 31 or 104. In certain embodiments, thecell-targeting molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more) identical to the amino acid sequence shown in any one of SEQ IDNOs: 34 or 106. In certain embodiments, the cell-targeting molecule ofthe present invention comprises, consists essentially of, or consists ofan amino acid sequence that is at least 85% (such as at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the aminoacid sequence shown in any one of SEQ ID NOs: 35 or 107. In certainembodiments, the cell-targeting molecule of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequenceshown in any one of SEQ ID NOs: 36 or 108.

Accordingly, in certain embodiments, the Shiga toxin effectorpolypeptides of the present invention comprise, consists essentially of,or consists of amino acid sequences having at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, overall sequence identity toa naturally occurring (e.g. a wild-type) Shiga toxin A Subunit orfragment thereof, such as, e.g., Shiga toxin A Subunit, such as SLT-1A(SEQ ID NO:1), StxA (SEQ ID NO:2), SLT-2A (SEQ ID NO:3), Stx1cA (SEQ IDNO:4), Stx1dA (SEQ ID NO:5), Stx1eA (SEQ ID NO:6), Stx2cA variant 1 (SEQID NO:7), Stx2cA variant 2 (SEQ ID NO:8), Stx2cA variant 3 (SEQ IDNO:9), Stx2cA variant 4 (SEQ ID NO: 10), Stx2cA variant 5 (SEQ ID NO:11), Stx2cA variant 6 (SEQ ID NO: 12), Stx2dA variant 1 (SEQ ID NO: 13),Stx2dA variant 2 (SEQ ID NO: 14), Stx2dA variant 3 (SEQ ID NO: 15),Stx2eA variant 1 (SEQ ID NO: 16), Stx2eA variant 2 (SEQ ID NO: 17),and/or Stx2fA (SED NO: 18), wherein the Shiga toxin effector polypeptide(1) comprises at least one embedded or inserted, heterologous T-cellepitope and at least one amino acid is disrupted in an endogenous,B-cell and/or CD4+ T-cell epitope region, and wherein the disruptedamino acid does not overlap with the embedded or inserted epitope; (2)comprises at least one embedded or inserted, heterologous T-cell epitopeand a disrupted furin-cleavage motif at the carboxy-terminus of a Shigatoxin A1 fragment derived region; or (3) comprises a disruptedfurin-cleavage motif at the carboxy-terminus of a Shiga toxin A1fragment derived region and comprises at least one amino acid isdisrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region,and wherein the disrupted amino acid does not overlap with the disruptedfurin-cleavage motif; or (4) comprises (i) at least one embedded orinserted, heterologous T-cell epitope, (ii) at least one amino acid isdisrupted in an endogenous, B-cell and/or CD4+ T-cell epitope region,wherein the disrupted amino acid does not overlap with the embedded orinserted epitope, and (iii) a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region. Asdescribed herein, fragments of the Shiga toxin A Subunit may comprise,consist essentially of, or consists of: (i) amino acids 75 to 251 of anyone of SEQ ID NOs: 1-6, 37, and 75-89; (ii) amino acids 1 to 241 of anyone of SEQ ID NOs: 1-18, 37, and 75-89; (iii) amino acids 1 to 251 ofany one of SEQ ID NOs: 1-6, 37 and 75-89; or (iv) amino acids 1 to 261of any one of SEQ ID NOs: 1-3. For example, the fragments of the Shigatoxin A Subunit may comprise, consist essentially of, or consists ofamino acids: (i) 75 to 251 of any one SEQ ID NOs: 75-89, (ii) 1 to 241of any one of SEQ ID NOs: 75-89, (iii) 1 to 251 of any one of SEQ IDNOs: 75-89, or (iv) 1 to 261 of any one of SEQ ID NOs: 1-3.

In certain embodiments of the Shiga toxin effector polypeptides of thepresent invention, one or more amino acid residues may be mutated,inserted, or deleted in order to increase the enzymatic activity of theShiga toxin effector polypeptide. In certain embodiments of the Shigatoxin effector polypeptides of the present invention, one or more aminoacid residues may be mutated or deleted in order to reduce or eliminatecatalytic and/or cytotoxic activity of the Shiga toxin effectorpolypeptide. For example, the catalytic and/or cytotoxic activity of theA Subunits of members of the Shiga toxin family may be diminished oreliminated by mutation or truncation.

The cytotoxicity of the A Subunits of members of the Shiga toxin familymay be altered, reduced, or eliminated by mutation and/or truncation.The positions labeled tyrosine-77, glutamate-167, arginine-170,tyrosine-114, and tryptophan-203 have been shown to be important for thecatalytic activity of Stx, Stx1, and Stx2 (Hovde C et al., Proc NatlAcad Sci USA 85: 2568-72 (1988); Deresiewicz R et al., Biochemistry 31:3272-80 (1992); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993);Ohmura M et al., Microb Pathog 15: 169-76 (1993); Cao C et al.,Microbiol Immunol 38: 441-7 (1994); Suhan M, Hovde C, Infect Immun 66:5252-9 (1998)). Mutating both glutamate-167 and arginine-170 eliminatedthe enzymatic activity of Slt-I A1 in a cell-free ribosome inactivationassay (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)). In anotherapproach using de novo expression of Slt-I A1 in the endoplasmicreticulum, mutating both glutamate-167 and arginine-170 eliminated Slt-IA1 fragment cytotoxicity at that expression level (LaPointe P et al., JBiol Chem 280: 23310-18 (2005)). A truncation analysis demonstrated thata fragment of StxA from residues 75 to 268 still retains significantenzymatic activity in vitro (Haddad J et al., J Bacteriol 175: 4970-8(1993)). A truncated fragment of Slt-I A1 containing residues 1-239displayed significant enzymatic activity in vitro and cytotoxicity by denovo expression in the cytosol (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). Expression of a Slt-I A1 fragment truncated toresidues 1-239 in the endoplasmic reticulum was not cytotoxic because itcould not retrotranslocate to the cytosol (LaPointe P et al., J BiolChem 280: 23310-18 (2005)).

The most critical residues for enzymatic activity and/or cytotoxicity inthe Shiga toxin A Subunits were mapped to the followingresidue-positions: asparagine-75, tyrosine-77, tyrosine-114,glutamate-167, arginine-170, arginine-176, and tryptophan-203 amongothers (Di R et al., Toxicon 57: 525-39 (2011)). In particular, adouble-mutant construct of Stx2A containing glutamate-E167-to-lysine andarginine-176-to-lysine mutations was completely inactivated; whereas,many single mutations in Stx1 and Stx2 showed a 10-fold reduction incytotoxicity. Further, truncation of Stx1A to 1-239 or 1-240 reduced itscytotoxicity, and similarly, truncation of Stx2A to a conservedhydrophobic residue reduced its cytotoxicity. The most critical residuesfor binding eukaryotic ribosomes and/or eukaryotic ribosome inhibitionin the Shiga toxin A Subunit have been mapped to the followingresidue-positions arginine-172, arginine-176, arginine-179,arginine-188, tyrosine-189, valine-191, and leucine-233 among others(McCluskey A et al., PLoS One 7: e31191 (2012). However, certainmodification may increase a Shiga toxin functional activity exhibited bya Shiga toxin effector polypeptide of the present invention. Forexample, mutating residue-position alanine-231 in Stx1A to glutamateincreased Stx1A’s enzymatic activity in vitro (Suhan M, Hovde C, InfectImmun 66: 5252-9 (1998)).

In certain embodiments of Shiga toxin effector polypeptides of thepresent invention derived from SLT-1A (SEQ ID NO:1) or StxA (SEQ IDNO:2), the one or more amino acid residues mutated include substitutionof the asparagine at position 75, tyrosine at position 77, tyrosine atposition 114, glutamate at position 167, arginine at position 170,arginine at position 176, and/or substitution of the tryptophan atposition 203. Examples of such substitutions will be known to theskilled worker based on the prior art, such as asparagine at position 75to alanine, tyrosine at position 77 to serine, substitution of thetyrosine at position 114 to serine, substitution of the glutamateposition 167 to glutamate, substitution of the arginine at position 170to alanine, substitution of the arginine at position 176 to lysine,substitution of the tryptophan at position 203 to alanine, and/orsubstitution of the alanine at 231 with glutamate. Other mutations whicheither enhance or reduce Shiga toxin enzymatic activity and/orcytotoxicity are within the scope of the invention and may be determinedusing well known techniques and assays disclosed herein.

In certain embodiments, the cell-targeting molecule of the presentinvention may be monovalent and/or monomeric. In certain embodiments,the cell-targeting molecule of the present invention may not bemultivalent and/or multimeric. As demonstrated by the Examples of theapplication, monovalent and/or monomeric forms of certain cell-targetingmolecules may exhibit low levels of toxicity when used in vivo whilestill exhibiting potent cytotoxic to HER2-expressing cells.

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention may optionally be conjugated to one or moreadditional agents, which may include therapeutic agents, diagnosticagents, and/or other additional exogenous materials known in the art,including such agents as described herein. In certain embodiments, theShiga toxin effector polypeptide or cell-targeting molecule of thepresent invention is PEGylated or albuminated, such as, e.g., to providede-immunization, disrupt furin-cleavage by masking the extended loopand/or the furin-cleavage motif at the carboxy-terminus of a Shiga toxinA1 fragment derived region, improve pharmacokinetic properties, and/orimprove immunogenicity (see e.g., Wang Q et al., Cancer Res 53: 4588-94(1993); Tsutsumi Y et al., Proc Natl Acad Sci USA 97: 8548-53 (2000);Buse J, El-Aneed A, Nanomed 5: 1237-60 (2010); Lim S et al., J ControlRelease 207-93 (2015)).

V. General Functions of the Cell-Targeting Molecules of the PresentInvention

The functional association of Shiga toxin effector polypeptides of thepresent invention with cell-targeting binding regions enables thecreation of cell-targeting molecules which selectively kill, inhibit thegrowth of, deliver exogenous material to, and/or detect specific celltypes. The properties of the Shiga toxin effector polypeptide of thepresent invention enable the creation of cell-targeting molecules withimproved therapeutic windows in chordates as compared to prior Shigatoxin effector polypeptides.

For certain embodiments, the cell-targeting molecule of the presentinvention provides, after administration to a chordate, one or more ofthe following: 1) potent and selective killing of targeted cells, e.g.,infected or malignant cells, at low administration doses, 2) linkagestability between the cell-targeting binding region and the Shiga toxineffector polypeptide region while the cell-targeting molecule is presentin extracellular spaces, 3) low levels of off-target cell deaths and/orunwanted tissue damage, and 4) cell-targeted delivery of heterologous,CD8+ T-cell epitopes for presentation by target cells in order toinitiate desirable, T-cell mediated, immune responses, such as, e.g.,the recruitment of CD8+ T-cells and the localized release of cytokinesat a tissue locus.

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention are useful in diverse applications involving,e.g., cell-killing; cell growth inhibition; intracellular, cargodelivery; biological information gathering; immune response stimulation,and/or remediation of a health condition. The Shiga toxin effectorpolypeptides of the present invention are useful as components ofvarious therapeutic and/or diagnostic molecules, such as, e.g.ligand-toxin fusions, immunotoxins, and/or immuno-conjugates. Thecell-targeting molecules of the present invention are useful astherapeutic and/or diagnostic molecules, such as, e.g., ascell-targeting, cytotoxic, therapeutic molecules; cell-targeting,nontoxic, delivery vehicles; and/or cell-targeting, diagnosticmolecules; for examples in applications involving the in vivo targetingof specific cell types for the diagnosis or treatment of a variety ofdiseases, including cancers, immune disorders, and microbial infections.

Depending on the embodiment, a Shiga toxin effector polypeptide orcell-targeting molecule of the present invention may have or provide oneor more of the following characteristics or functionalities: (1)de-immunization, (2) protease-cleavage resistance, (3) potentcytotoxicity at certain concentrations, (4) intracellular delivery of acargo consisting of an additional material (e.g. a heterologous, T-cellepitope), (4) selective cytotoxicity, (6) low off-target toxicity inmulticellular organisms at certain doses or dosages, (7) delivery of aheterologous, T-cell epitope to the MHC class I presentation pathway ofa target cell, and/or (8) stimulation of CD8+ T-cell immune response(s).Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are multifunctionalbecause the molecules have two or more of the characteristics orfunctionalities described herein. Certain further embodiments of thecell-targeting molecules of the present invention provide all of theaforementioned characteristics and functionalities in a single molecule.

The associating, coupling, and/or linking of a cell-targeting bindingregion(s) with a Shiga toxin effector polypeptide(s) of the presentinvention enables the engineering of cell-targeting molecules with Shigatoxin function(s) that can produce less adverse effects afteradministration at certain doses or dosages to a multicellular organismsuch as a mammal. Non-limiting examples of adverse effects includeoff-target toxicities, untargeted cytotoxicities, and/or unwanted immuneresponses. Certain embodiments of the Shiga toxin effector polypeptidesand cell-targeting molecules of the present invention are particularlyuseful in applications involving administration of a Shiga toxineffector polypeptide and/or cell-targeting molecule to a chordatebecause of functional properties, such as, e.g., de-immunization,reduced off-target toxicities, and/or targeted stimulation of desirableimmune responses such as via cell-surface presentation of acell-targeting molecule delivered, CD8+ T-cell epitope.

In certain embodiments, the cell-targeting molecules of the presentinvention are capable of binding extracellular target biomoleculesassociated with the cell surface of particular cell types and enteringthose cells. Once internalized within a targeted cell type, certainembodiments of the cell-targeting molecules of the invention are capableof routing an enzymatically active, cytotoxic, Shiga toxin effectorpolypeptide fragment into the cytosol of the target cell and eventuallykilling the cell. Alternatively, nontoxic or reduced-toxicity variantsof the cell-targeting molecules of the present invention may be used todeliver additional exogenous materials into target cells, such asepitopes, peptides, proteins, polynucleotides, and detection promotingagents. This system is modular, in that any number of diverse bindingregions can be used to target a Shiga toxin effector polypeptide of thepresent invention to various, diverse cell types.

A. De-Immunization for Applications Involving Administration to aChordate

The de-immunization of the Shiga toxin effector polypeptides of thepresent invention is accomplished by engineering disruptions of one ormore, endogenous, B-cell and/or CD4+ T-cell epitopes regions of a Shigatoxin A Subunit or Shiga toxin effector polypeptide, including viamutation and/or truncation or via the conjugation of a covalently-linkedchemical structure. Because B-cell epitopes often coincide or overlapwith epitopes of mature CD4+ T-cells, the disruption of an endogenous,B-cell epitope region often simultaneously disrupts an endogenous, CD4+T-cell epitope or vice versa.

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are de-immunized withrespect to one or more B-cell and/or CD4+ T-cell epitopes meaning thatthese molecules exhibit reduced antigenic and/or immunogenic potentialas compared to prior, Shiga toxin effector polypeptides andcell-targeting molecules lacking identical disruptions to the sameB-cell and/or CD4+ T-cell epitope or epitope regions and/or lacking anydisruption to the same B-cell and/or CD4+ T-cell epitope(s) or epitoperegion(s). Certain further embodiments exhibit potent if not wild-typelevels of Shiga toxin A Subunit catalytic domain dependent cytotoxicitydespite the presence of multiple mutations providing the de-immunizedproperty. The de-immunized, Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are useful forapplications involving the parenteral administration of a Shiga toxineffector polypeptide and/or cell-targeting molecule to a chordate suchas, e.g., a mammal, amphibian, bird, fish, reptiles, or shark, becauseof the reduced likelihood of producing undesirable immune responsesinvoked by the administrated molecule.

The various de-immunized, Shiga toxin effector polypeptides of thepresent invention might differ in their antigenicity profiles whenadministered to various chordate species, but all the de-immunizedpolypeptides of the invention exhibit reduced antigenicity and/orimmunogenicity in at least one organism as measured by at least onequantitative assay. In particular, certain embodiments of thecell-targeting molecules of the present invention are de-immunized withrespect to a mammalian recipient, such as, e.g., the molecule invokeslower quantities and/or frequencies of “anti-cell-targeting molecule”antibodies when administered to that mammal as compared to a referencemolecule (e.g. a related cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment). In addition, Shiga toxin effector polypeptidesof the present invention having disruptions of multiple, endogenous,epitope regions are expected to more greatly reduced the probability ofthe occurrence of undesirable immune responses in a chordate recipientof such a polypeptide.

For certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, the de-immunizationproperty(ies) is a result of the structural change(s) which include thedisrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxinA1 fragment derived region.

For certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, the de-immunizationproperty(ies) is a result of the structural change(s) which include theembedding and/or inserting of a T-cell epitope which disrupts anendogenous, B-cell and/or CD4+ T-cell epitope region.

For certain embodiments, the desired biological function(s) of theparental, Shiga toxin polypeptide from which the de-immunized, Shigatoxin effector polypeptide was derived are preserved, such as, e.g., theShiga toxin A Subunit functions of promoting cellular internalization,directing intracellular routing, and potent cytotoxicity. Preservationrefers to the retention of a minimal level of activity as describedherein.

B. Reduced Protease-Cleavage Sensitivity

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention exhibit reducedprotease-cleavage sensitivity as compared to related moleculescomprising wild-type, Shiga toxin A1 fragment regions. Certain furtherembodiments exhibit potent if not optimal, Shiga toxin A Subunitcatalytic domain dependent cytotoxicity despite this reducedprotease-cleavage sensitivity and lack of a canonical furin-cleavageevent within an intoxicated cell.

Certain embodiments of the protease-cleavage resistant, cell-targetingmolecules of the present invention (i.e. a cell-targeting moleculecomprising a Shiga toxin effector polypeptide comprising a disruptedfurin-cleavage motif at the carboxy-terminus of its Shiga toxin A1fragment region) exhibit improved in vivo tolerability as compared torelated molecules comprising a wild-type, Shiga toxin A1 fragmentregion. Certain further embodiments exhibit potent if not optimal, Shigatoxin A Subunit catalytic domain dependent cytotoxicity despite thisreduced protease-cleavage sensitivity and lack of a canonicalfurin-cleavage event within an intoxicated cell.

Previously, it was believed that cytotoxic, Shiga toxin A Subunitconstructs comprising Shiga toxin A1 fragment catalytic regions mustmaintain or somehow compensate for the naturally occurring proteolyticprocessing by furin within intoxicated cells in order to preserve theShiga toxin’s natural adaptations for efficient and potent cytotoxicity.It was unexpectedly discovered that the furin cleavage event was notrequired for potent cytotoxicity because potent Shiga toxin cytotoxicityat the level of a wild-type Shiga toxin control construct was achievedin the absence of any furin cleavage event at the carboxy-terminus ofthe Shiga toxin A1 fragment despite the presence of a carboxy-terminalmoiety (see e.g. WO 2015/191764; WO 2016/196344). The lack of afurin-cleavage event within the intoxicated cell may prevent theefficient liberation of a Shiga toxin A1 fragment-like region and, thus,result in the continued linkage of a relatively large moiety (e.g.greater than 28 kDa in size) to the Shiga toxin A1 fragment region.However despite this possibility, potent, Shiga toxin cytotoxicity wasachieved with furin-cleavage deficient constructs comprising a Shigatoxin effector polypeptide region and lacking any known compensatoryfeature(s), such as, e.g., providing intracellular cleavage proximal tothe carboxy-terminus of a Shiga toxin A1 fragment derived region (seee.g. WO 2015/191764; WO 2016/196344).

This suggests that the persistence and/or inefficient release of arelatively large, molecular moiety linked to the A1 fragment region didnot necessarily reduce the potency of Shiga toxin cytotoxicity. This wassurprising because the optimal Shiga toxin intoxication process wasthought to require liberation of the Shiga toxin A1 fragments from allother large molecular moieties to efficiently retrotranslocate liberatedA1 fragments from the endoplasmic reticulum to the cytosol where the A1fragments can form an enzymatically active structure that catalyticallyinactivates the intoxicated cell’s ribosomes. In particular, thepersistence and/or inefficient release of a relatively large molecularmoiety covering the carboxy-terminus of the Shiga toxin A1 fragment wasexpected to interfere with the Shiga toxin A1 fragment’s naturalmechanism of efficiently gaining access to the cytosol, which involvesthe exposure of the A1 fragment’s, hydrophobic, carboxy-terminal domainand recognition of this domain by the ERAD system (see Di R et al.,Toxicon 57: 525-39 (2011); Li S et al., PLoS One 7: e41119 (2012)).

The lack of an intoxicated-cell-mediated, furin-cleavage event for amolecule comprising a Shiga toxin A Subunit derivative may behypothetically compensated for. Non-limiting examples of potential,compensatory approaches include 1) terminating one carboxy-terminus ofthe construct with the carboxy-terminus of a Shiga toxin A1fragment-like polypeptide region, 2) producing the Shiga toxin derivedconstruct such that the Shiga toxin A Subunit polypeptide is alreadynicked near the carboxy-terminus of its Shiga toxin A1 fragment-likepolypeptide, 3) engineering a heterologous and/or ectopic protease sitethat can functionally substitute for the lack of the native, Shigatoxin, furin-cleavage event, and 4) a combination of approach 3 and 4.

In the first approach, the carboxy-terminus of the Shiga toxin A1fragment-like polypeptide is not covered by any carboxy-terminal moiety,and, thus, the carboxy-terminus of the Shiga toxin A1 fragment-likepolypeptide is permanently exposed for recognition by the ERAD machineryin the endoplasmic reticulum. In the last three approaches, the Shigatoxin A1 fragment-like polypeptide can be designed to intracellularlydissociate from one or more other components of the construct by thetime the molecule reaches the endoplasmic reticulum of an intoxicatedcell such that in the endoplasmic reticulum the carboxy-terminus of theShiga toxin A1 fragment-like polypeptide becomes exposed for recognitionby the ERAD machinery. For example, a cytotoxic molecule comprising aShiga toxin A Subunit effector polypeptide could be pretreated with aprotease to nick the polypeptide region near the carboxy terminus of theA1 fragment-like region prior to contacting a target cell.Alternatively, the cytotoxic molecule could be engineered to comprise aprotease site which is cleaved by an intracellular protease of thetarget cell.

These hypothetical approaches for designing Shiga toxin A Subuniteffector polypeptides which compensate for the lack of anintoxicated-cell-mediated, furin-cleavage event may significantly alterthe efficiency and potency of cytotoxicity as compared to a wild-typeShiga holotoxin or Shiga toxin A Subunit construct comprising onlywild-type sequences which include the optimal, naturally occurring,furin-cleavage site. For example, currently no compensatory approachrelying on a target cell endoprotease other than furin is known whichcan provide fully compensatory cytotoxicity equivalent to furin cleavageand alternative cellular proteases to furin like calpains have beenshown to be less efficient in facilitating Shiga toxin cytotoxicity(Garred O et al., Exp Cell Res 218: 39-49 (1995); Garred O et al., JBiol Chem 270: 10817-21 (1995); Kurmanova A et al., Biochem Biophys ResCommun 357: 144-9 (2007)).

The present invention provides furin-cleavage resistant Shiga toxin ASubunit effector polypeptides which are potently cytotoxic, whether dueto compensation for a lack of a furin cleavage event within theintoxicated cell or due to some unexplained reason. Certaincell-targeting molecules of the present invention are at least asefficiently and potently cytotoxic as cell-targeting moleculescomprising protease-cleavage sensitive, wild-type Shiga toxin effectorpolypeptide regions (see e.g. WO 2016/196344).

C. Improved Stability and In Vivo Tolerability

In certain embodiments, the molecules of the present invention (e.g.cell-targeting molecules of the invention) exhibit increased stabilityand/or improved in vivo tolerability as compared to more furin-cleavagesensitive analogs and/or less de-immunized analogs (an analog being aclosely related molecule lacking one or more structural features of thepresent invention).

The increased stability of a cell-targeting molecule compared to areference molecule can be exhibited in vitro and/or in vivo. Thestability of a therapeutic or diagnostic molecule over time is animportant feature and can affect for which applications the molecule maybe practically employed. Molecular stability includes in vitro and invivo, such as, e.g., stability within an organism after administrationand during storage over a range of temperatures and concentrations. Forcertain immunotoxins or ligand-toxin fusions, the stability of thelinkage between the toxin and other components can affect the amount ofnon-specific toxicity caused by the presence and/or quantity ofuntargeted toxin over time within the organism.

Certain cell-targeting molecules of the present invention exhibitreduced non-specific toxicity in vivo, manifested as increased in vivotolerability as compared to more protease-cleavage sensitive variants.In vivo tolerability can be determined by the skilled worker usingtechniques known in the art and/or described herein. In addition toassessing in vivo tolerability using mortality, signs of morbidity maybe used for assessing in vivo tolerability, such as, e.g., aspects ofbody weight, physical appearance, measureable clinical signs, unprovokedbehavior, and responses to external stimuli (see e.g. Morton D,Griffiths P, Vet Rec 116: 431-43 (1985); Montgomery C, Cancer Bull 42:230-7 (1990); Ullman-Culleré M, Foltz C, Lab Anim Sci 49: 319-23 (1999);Clingerman K, Summers L, J Am Assoc Lab Anim Sci 51: 31-6 (2012)).Euthanasia may be used in response to signs of morbidity and/ormorbundity and, thus, create a mortality time-point. For example, adecrease in body weight of 15-20% in 2-3 days can be used as a sign ofmorbidity in rodents and as a justification for euthanization (see e.g.Institute of Laboratory Animal Research 2011. Guide for the care and useof laboratory animals, 8th ed., Washington, DC, U.S.: National AcademiesPress).

The improved in vivo tolerability observed for exemplary, cell-targetingmolecules of the present invention as compared to more furin-cleavagesensitive analogs suggests that much higher doses of thesecell-targeting molecules of the invention may be safely administered tomammals as compared to the doses of related molecules comprising afurin-cleavage sensitive, Shiga toxin effector polypeptide region.Certain cell-targeting molecules of the invention might exhibit reducednon-specific toxicity as compared to more protease sensitive variantsbecause the protease resistance serves to protect and preserve thelinkage between the Shiga toxin effector component and thecell-targeting moiety component.

In addition, in vivo tolerability for cell-targeting molecules of thepresent invention may be related to the de-immunization properties of agiven cell-targeting molecule. Thus, higher doses of such de-immunized,cell-targeting molecules of the invention may be safely administered tomammals as compared to the doses of related molecules comprising an“un-de-immunized” or less de-immunized, Shiga toxin effector polypeptide(e.g. a wild-type Shiga toxin A1 fragment).

In addition, certain molecules of the invention exhibit increasedhalf-lives, both in vitro and/or in vivo, as compared to moreprotease-cleavage sensitive variants. Molecular stability can be assayedby determining the half-life of a molecule of interest with regard tothe association of its components. Certain embodiments of the moleculesof the invention will have longer half-lives as compared tofurin-cleavage sensitive variants, especially with regard to thecontinued association of the Shiga toxin effector polypeptide componentand one or more other components. For example, certain embodiments ofthe molecules of the invention will have longer half-lives with regardto the continued association of the Shiga toxin effector polypeptidecomponent and another component, e.g. a cell-targeting binding region,as compared to a furin-cleavage sensitive variant wherein thefurin-cleavage sensitive site(s) lies between those two components.

D. Cell-Kill via Shiga Toxin A Subunit Cytotoxicity

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are cytotoxic. Certainfurther embodiments of the cell-targeting molecules of the presentinvention are cytotoxic only due to the presence of one or more Shigatoxin effector polypeptide components. The A Subunits of members of theShiga toxin family each comprise an enzymatically active polypeptideregion capable of killing a eukaryotic cell once in the cell’s cytosol.Because members of the Shiga toxin family are adapted to killingeukaryotic cells, molecules derived from Shiga toxins, such as, e.g.,molecules comprising certain embodiments of the Shiga toxin effectorpolypeptides of the present invention can exhibit potent cell-killactivities.

For certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the binding region of thecell-targeting molecule (e.g. a target positive cell), thecell-targeting molecule is capable of causing death of the cell. Forcertain further embodiments, the CD₅₀ value of the cell-targetingmolecule is less than 5, 2.5, 1, 0.5, or 0.25 nM, which is vastly morepotent than an untargeted, wild-type, Shiga toxin effector polypeptide(e.g. SEQ ID NOs: 1-18).

Cell-kill may be accomplished using a molecule of the present inventionunder varied conditions of target cells, such as, e.g., an ex vivomanipulated target cell, a target cell cultured in vitro, a target cellwithin a tissue sample cultured in vitro, or a target cell in an in vivosetting like within a multicellular organism.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention comprise (1) ade-immunized, Shiga toxin effector sub-region, (2) a protease-cleavageresistant region near the carboxy-terminus of a Shiga toxin A1 fragmentderived region, (3) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif; and/or (4) a heterologous, T-cellepitope embedded or inserted region; however, for certain furtherembodiments, these structural modifications do not significantly alterthe potency of Shiga toxin cytotoxicity as compared to a referencemolecules comprising a wild-type Shiga toxin A Subunit polypeptide, suchas, e.g., a wild-type Shiga toxin A1 fragment. Thus, Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention which are de-immunized, protease cleavage resistant, and/orcarrying embedded or inserted, heterologous, epitopes can maintainpotent cytotoxicity while providing one or more various otherfunctionalities or properties.

Already cytotoxic cell-targeting molecules comprising Shiga toxineffector polypeptides may be engineered by the skilled worker using theinformation and methods provided herein to be more cytotoxic and/or tohave redundant, backup cytotoxicities operating via completely differentmechanisms. These multiple cytotoxic mechanisms may complement eachother by their diversity of functions (such as by providing potentkilling via two mechanisms of cell-killing, direct and indirect, as wellas mechanisms of immuno-stimulation to the local area), redundantlybackup each other (such as by providing one cell-killing mechanism inthe absence of the other mechanisms-like if a target cell is resistantto or acquires some immunity to a subset of previously activemechanisms), and/or protect against developed resistance (by limitingresistance to the less probable situation of the malignant or infectedcell blocking multiple, different cell-killing mechanismssimultaneously).

E. Delivery of a T-Cell Epitope for MHC Class I Presentation on a CellSurface

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention comprise a T-cellepitope, which enables the engineering of “T-cell epitope delivering”molecules with virtually unlimited choices of epitope-peptide cargos fordelivery and cell-surface presentation by a nucleated, chordate cell.For certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are each capable ofdelivering one or more T-cell epitopes, associated with the Shiga toxineffector polypeptides and/or cell-targeting molecules, to the proteasomeof a cell. The delivered T-cell epitope are then proteolytic processedand presented by the MHC class I pathway on the surface of the cell. Byengineering MHC class I epitopes into cell-targeting molecules, thetargeted delivery and presentation of immuno-stimulatory antigens may beaccomplished in order to harness and direct a beneficial function(s) ofa chordate immune system.

For certain embodiments, the cell-targeting molecule of the presentinvention is capable of delivering a T-cell epitope to a MHC class Imolecule of a cell for cell-surface presentation. In certainembodiments, the Shiga toxin effector polypeptide or cell-targetingmolecule of the present invention comprises a heterologous, T-cellepitope, whether as an additional exogenous material or embedded orinserted within a Shiga toxin effector polypeptide. For certain furtherembodiments, the Shiga toxin effector polypeptide or cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted T-cell epitope to a MHC class I molecule for cell-surfacepresentation.

For certain embodiments, the Shiga toxin effector polypeptide of thepresent invention is capable of delivering a T-cell epitope, which isembedded or inserted in the Shiga toxin effector polypeptide, to a MHCclass I molecule of a cell in which the Shiga toxin effector polypeptideis present for presentation of the T-cell epitope by the MHC class Imolecule on a surface of the cell. For certain further embodiments, theT-cell epitope is a heterologous, T-cell epitope. For certain furtherembodiments, the T-cell epitope functions as CD8+ T-cell epitope,whether already known or identified in the future using methods whichare currently routine to the skilled worker.

For certain embodiments, the cell-targeting molecule of the presentinvention is capable of delivering a T-cell epitope, which is associatedwith the cell-targeting molecule, to a MHC class I molecule of a cellfor presentation of the T-cell epitope by the MHC class I molecule on asurface of the cell. For certain further embodiments, the T-cell epitopeis a heterologous, T-cell epitope which is embedded or inserted in theShiga toxin effector polypeptide. For certain further embodiments, theT-cell epitope functions as CD8+ T-cell epitope, whether already knownor identified in the future using methods which are currently routine tothe skilled worker.

For certain embodiments, upon contacting a cell with the cell-targetingmolecule of the present invention, the cell-targeting molecule iscapable of delivering a T-cell epitope-peptide, which is associated withthe cell-targeting molecule, to a MHC class I molecule of the cell forpresentation of the T-cell epitope-peptide by the MHC class I moleculeon a surface of the cell. For certain further embodiments, the T-cellepitope-peptide is a heterologous epitope which is embedded or insertedin a Shiga toxin effector polypeptide. For certain further embodiments,the T-cell epitope-peptide functions as CD8+ T-cell epitope, whetheralready known or identified in the future using methods which arecurrently routine to the skilled worker.

The addition of a heterologous epitope into or presence of aheterologous epitope in a cell-targeting molecule of the presentinvention, whether as an additional exogenous material or embedded orinserted within a Shiga toxin effector polypeptide, enables methods ofusing such cell-targeting molecules for the cell-targeted delivery of achosen epitope for cell-surface presentation by a nucleated, target cellwithin a chordate.

One function of certain, CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention is the delivery of one or more T-cell epitope-peptides to aMHC class I molecule for MHC class I presentation by a cell. Delivery ofexogenous, T-cell epitope-peptides to the MHC class I system of a targetcell can be used to induce the target cell to present the T-cellepitope-peptide in association with MHC class I molecules on the cellsurface, which subsequently leads to the activation of CD8+ effectorT-cells to attack the target cell.

The skilled worker, using techniques known in the art, can associate,couple, and/or link certain, Shiga toxin effector polypeptides of thepresent invention to various other cell-targeting binding region tocreate cell-targeting molecules of the present invention which targetspecific, extracellular, target biomolecules physically coupled to cellsand promote target-cell internalization of these cell-targetingmolecules. All nucleated vertebrate cells are believed to be capable ofpresenting intracellular epitopes using the MHC class I system. Thus,extracellular target biomolecules of the cell-targeting molecules of theinvention may in principle target any nucleated vertebrate cell forT-cell epitope delivery to a MHC class I presentation pathway of such acell.

The epitope-delivering functions of the Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention canbe detected and monitored by a variety of standard methods known in theart to the skilled worker and/or described herein. For example, theability of cell-targeting molecules of the present invention to delivera T-cell epitope-peptide and drive presentation of the epitope-peptideby the MHC class I system of target cells may be investigated usingvarious in vitro and in vivo assays, including, e.g., the directdetection/visualization of MHC class I/peptide complexes, measurement ofbinding affinities for the heterologous, T-cell epitope-peptide to MHCclass I molecules, and/or measurement of functional consequences of MHCclass I-peptide complex presentation on target cells by monitoringcytotoxic T-lymphocyte (CTL) responses (see e.g. Examples, infra).

Certain assays to monitor this function of the polypeptides andmolecules of the present invention involve the direct detection of aspecific MHC class I/peptide antigen complex in vitro or ex vivo. Commonmethods for direct visualization and quantitation of peptide-MHC class Icomplexes involve various immuno-detection reagents known to the skilledworker. For example, specific monoclonal antibodies can be developed torecognize a particular MHC/class I/peptide antigen complex. Similarly,soluble, multimeric T cell receptors, such as the TCR-STAR reagents(Altor Bioscience Corp., Mirmar, FL, U.S.) can be used to directlyvisualize or quantitate specific MHC I/antigen complexes (Zhu X et al.,J lmmunol 176: 3223-32 (2006)). These specific mAbs or soluble,multimeric T-cell receptors may be used with various detection methods,including, e.g. immunohistochemistry, flow cytometry, and enzyme-linkedimmuno assay (ELISA).

An alternative method for direct identification and quantification ofMHC I/peptide complexes involves mass spectrometry analyses, such as,e.g., the ProPresent Antigen Presentation Assay (ProImmune, Inc.,Sarasota, FL, U.S.) in which peptide-MCH class I complexes are extractedfrom the surfaces of cells, then the peptides are purified andidentified by sequencing mass spectrometry (Falk K et al., Nature 351:290-6 (1991)).

In certain assays to monitor the T-cell epitope delivery and MHC class Ipresentation function of the polypeptides and molecules of the presentinvention involve computational and/or experimental methods to monitorMHC class I and peptide binding and stability. Several software programsare available for use by the skilled worker for predicting the bindingresponses of peptides to MHC class I alleles, such as, e.g., The ImmuneEpitope Database and Analysis Resource (IEDB) Analysis Resource MHC-Ibinding prediction Consensus tool (Kim Y et al., Nucleic Acid Res 40:W525-30 (2012). Several experimental assays have been routinely applied,such as, e.g., cell surface binding assays and/or surface plasmonresonance assays to quantify and/or compare binding kinetics (Miles K etal., MolImmunol 48: 728-32 (2011)). Additionally, other MHC-peptidebinding assays based on a measure of the ability of a peptide tostabilize the ternary MHC-peptide complex for a given MHC class Iallele, as a comparison to known controls, have been developed (e.g.,MHC-peptide binding assay from ProImmmune, Inc.).

Alternatively, measurements of the consequence of MHC class I/peptideantigen complex presentation on the cell surface can be performed bymonitoring the cytotoxic T-cell (CTL) response to the specific complex.These measurements by include direct labeling of the CTLs with MHC classI tetramer or pentamer reagents. Tetramers or pentamers bind directly toT cell receptors of a particular specificity, determined by the MajorHistocompatibility Complex (MHC) allele and peptide complex.Additionally, the quantification of released cytokines, such asinterferon gamma or interleukins by ELISA or enzyme-linked immunospot(ELIspot) is commonly assayed to identify specific CTL responses. Thecytotoxic capacity of CTL can be measured using a number of assays,including the classical 51 Chromium (Cr) release assay or alternativenon-radioactive cytotoxicity assays (e.g., CytoTox96® non-radioactivekits and CellTox™ CellTiter-GLO® kits available from Promega Corp.,Madison, WI, U.S.), Granzyme B ELISpot, Caspase Activity Assays orLAMP-1 translocation flow cytometric assays. To specifically monitor thekilling of target cells, carboxyfluorescein diacetate succinimidyl ester(CFSE) can be used to easily and quickly label a cell population ofinterest for in vitro or in vivo investigation to monitor killing ofepitope specific CSFE labeled target cells (Durward M et al., J Vis Exp45 pii 2250 (2010)).

In vivo responses to MHC class I presentation can be followed byadministering a MHC class I/antigen promoting agent (e.g., a peptide,protein or inactivated/attenuated virus vaccine) followed by challengewith an active agent (e.g. a virus) and monitoring responses to thatagent, typically in comparison with unvaccinated controls. Ex vivosamples can be monitored for CTL activity with methods similar to thosedescribed previously (e.g. CTL cytotoxicity assays and quantification ofcytokine release).

HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicatedcells after lysis using immune affinity (e.g., an anti-MHC antibody“pulldown” purification) and the associated peptides (i.e., the peptidespresented by the isolated MHC molecules) are recovered from the purifiedcomplexes. The recovered peptides are analyzed by sequencing massspectrometry. The mass spectrometry data is compared against a proteindatabase library consisting of the sequence of the exogenous (non-self)peptide (T-cell epitope X) and the international protein index forhumans (representing “self” or non-immunogenic peptides). The peptidesare ranked by significance according to a probability database. Alldetected antigenic (non-self) peptide sequences are listed. The data isverified by searching against a scrambled decoy database to reduce falsehits (see e.g. Ma B, Johnson R, Mol Cell Proteomics 11: 0111.014902(2012)). The results will demonstrate that peptides from the T-cellepitope X are presented in MHC complexes on the surface of intoxicatedtarget cells.

The set of presented peptide-antigen-MHC complexes can vary betweencells due to the antigen-specific HLA molecules expressed. T-cells canthen recognize specific peptide-antigen-MHC complexes displayed on acell surface using different TCR molecules with differentantigen-specificities.

Because multiple T-cell epitopes may be delivered by a cell-targetingmolecule of the invention, such as, e.g., by embedding two or moredifferent T-cell epitopes in a single proteasome delivering effectorpolypeptide, a single cell-targeting molecule of the invention may beeffective chordates of the same species with different MHC classvariants, such as, e.g., in humans with different HLA alleles. This mayallow for the combining within a single molecule of different T-cellepitopes with different effectiveness in different sub-populations ofsubjects based on MHC complex protein diversity and polymorphisms. Forexample, human MHC complex proteins, HLA proteins, vary among humansbased on genetic ancestry, e.g. African (sub-Saharan), Amerindian,Caucasoid, Mongoloid, New Guinean and Australian, or Pacific islander.

The applications involving the T-cell epitope delivering polypeptidesand molecules of the present invention are vast. Every nucleated cell ina mammalian organism may be capable of MHC class I pathway presentationof immunogenic, T-cell epitope-peptides on their cell outer surfacescomplexed to MHC class I molecules. In addition, the sensitivity ofT-cell epitope recognition is so exquisite that only a few MHC-I peptidecomplexes are required to be presented to result in an immune response,e.g., even presentation of a single complex can be sufficient forrecognition by an effector T-cell (Sykulev Y et al., Immunity 4: 565-71(1996)).

The activation of T-cell responses are desired characteristics ofcertain anti-cancer, anti-neoplastic, anti-tumor, and/or anti-microbialbiologic drugs to stimulate the patient’s own immune system towardtargeted cells. Activation of a robust and strong T-cell response isalso a desired characteristic of many vaccines. The presentation of aT-cell epitope by a target cell within an organism can lead to theactivation of robust immune responses to a target cell and/or itsgeneral locale within an organism. Thus, the targeted delivery of aT-cell epitope for presentation may be utilized for as a mechanism foractivating T-cell responses during a therapeutic regime.

The presentation of a T-cell immunogenic epitope-peptide by the MHCclass I system targets the presenting cell for killing by CTL-mediatedlysis and also triggers immune stimulation in the localmicroenvironment. By engineering immunogenic epitope sequences withinShiga toxin effector polypeptide components of target-cell-internalizingtherapeutic molecules, the targeted delivery and presentation ofimmuno-stimulatory antigens may be accomplished. The presentation ofimmuno-stimulatory non-self antigens, such as e.g. known viral antigenswith high immunogenicity, by target cells signals to other immune cellsto destroy the target cells as well as to recruit more immune cells tothe area.

The presentation of an immunogenic, T-cell epitope-peptide by the MHCclass I complex targets the presenting cell for killing by CTL-mediatedcytolysis. The presentation by targeted cells of immuno-stimulatorynon-self antigens, such as, e.g., known viral epitope-peptides with highimmunogenicity, can signal to other immune cells to destroy the targetcells and recruit more immune cells to the target cell site within achordate.

Thus, already cytotoxic molecules, such as e.g. therapeutic orpotentially therapeutic molecules comprising Shiga toxin effectorpolypeptides, may be engineered using methods of the present inventioninto more cytotoxic molecules and/or to have an additional cytotoxicmechanism operating via delivery of a T-cell epitope, presentation, andstimulation of effector T-cells. These multiple cytotoxic mechanisms maycomplement each other (such as by providing both directtarget-cell-killing and indirect (CTL-mediated) cell-killing,redundantly backup each other (such as by providing one mechanism ofcell-killing in the absence of the other), and/or protect against thedevelopment of therapeutic resistance (by limiting resistance to theless probable situation of the malignant or infected cell evolving toblock two different cell-killing mechanisms simultaneously).

In addition, a cytotoxic molecule comprising a Shiga toxin effectorpolypeptide region that exhibits catalytic-based cytotoxicity may beengineered by the skilled worker using routine methods intoenzymatically inactive variants. For example, the cytotoxic Shiga toxineffector polypeptide component of a cytotoxic molecule may be conferredwith reduced activity and/or rendered inactive by the introduction ofone or mutations and/or truncations such that the resulting molecule canstill be cytotoxic via its ability to deliver a T-cell epitope to theMHC class I system of a target cell and subsequent presentation to thesurface of the target cell. In another example, a T-cell epitope may beinserted or embedded into a Shiga toxin effector polypeptide such thatthe Shiga toxin effector polypeptide is inactivated by the added epitope(see e.g. WO 2015/113005). This approach removes one cytotoxic mechanismwhile retaining or adding another and may also provide a moleculecapable of exhibiting immuno-stimulation to the local area of a targetcell(s) within an organism via delivered T-cell epitope presentation or“antigen seeding.” Furthermore, non-cytotoxic variants of thecell-targeting molecules of the present invention which compriseembedded or inserted, heterologous, T-cell epitopes may be useful inapplications involving immune-stimulation within a chordate and/orlabeling of target cells within a chordate with MHC class I moleculedisplayed epitopes.

The ability to deliver a T-cell epitope of certain Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention maybe accomplished under varied conditions and in the presence ofnon-targeted bystander cells, such as, e.g., an ex vivo manipulatedtarget cell, a target cell cultured in vitro, a target cell within atissue sample cultured in vitro, or a target cell in an in vivo settinglike within a multicellular organism.

F. Cell-Kill via Targeted Cytotoxicity and/or Engagement of CytotoxicT-Cells

For certain embodiments, the cell-targeting molecule of the presentinvention can provide 1) delivery of a T-cell epitope for MHC class Ipresentation by a target cell and/or 2) potent cytotoxicity. For certainembodiments of the cell-targeting molecules of the present invention,upon contacting a cell physically coupled with an extracellular targetbiomolecule of the cell-targeting binding region, the cell-targetingmolecule of the invention is capable of causing death of the cell. Themechanism of cell-kill may be direct, e.g. via the enzymatic activity ofa toxin effector polypeptide region, or indirect via CTL-mediatedcytolysis.

1. Indirect Cell-Kill via T-Cell Epitope Delivery and MHC Class IPresentation

Certain embodiments of the cell-targeting molecules of the presentinvention are cytotoxic because they comprise a CD8+ T-cell epitopecapable of being delivered to the MHC class I presentation pathway of atarget cell and presented on a cellular surface of the target cell. Forexample, T-cell epitope delivering, CD8+ T-cell hyper-immunized, Shigatoxin effector polypeptides of the present invention, with or withoutendogenous epitope de-immunization, may be used as components ofcell-targeting molecules for applications involving indirectcell-killing.

In certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the cell-targeting binding region,the cell-targeting molecule of the invention is capable of indirectlycausing the death of the cell, such as, e.g., via the presentation ofone or more T-cell epitopes by the target cell and the subsequentrecruitment of CTLs which kill the target cell.

The presentation of an antigenic peptide complexed with a MHC class Imolecule by a cell sensitizes the presenting cell to targeted killing bycytotoxic T-cells (CTLs) via the induction of apoptosis, lysis, and/ornecrosis. In addition, the CTLs which recognize the target cell mayrelease immuno-stimulatory cytokines, such as, e.g., interferon gamma(IFN-gamma), tumor necrosis factor alpha (TNF), macrophage inflammatoryprotein-1 beta (MIP-1beta), and interleukins such as IL-17, IL-4, andIL-22. Furthermore, CTLs activated by recognition of a presented epitopemay indiscriminately kill other cells proximal to the presenting cellregardless of the peptide-MHC class I complex repertoire presented bythose proximal cells (Wiedemann A et al., Proc Natl Acad Sci USA 103:10985-90 (2006)).

Because of MHC allele diversity within different species, acell-targeting molecule of the present invention comprising only asingle epitope may exhibit varied effectiveness to different patients orsubjects of the same species. However, certain embodiments of thecell-targeting molecules of the present invention may each comprisemultiple, T-cell epitopes that are capable of being delivered to the MHCclass I system of a target cell simultaneously. Thus, for certainembodiments of the cell-targeting molecules of the present invention, acell-targeting molecule is used to treat different subjects withconsiderable differences in their MHC molecules’ epitope-peptide bindingaffinities (i.e. considerable differences in their MHC alleles and/orMHC genotypes). In addition, certain embodiments of the cell-targetingmolecules of the present invention reduce or prevent target celladaptations to escape killing (e.g. a target cancer cell mutating toescape therapeutic effectiveness or “mutant escape”) by using multiplecell-killing mechanisms simultaneously (e.g. direct killing and indirectkilling via multiple different T-cell epitopes simultaneously).

2. Direct Cell-Kill via Cell-Targeted, Shiga Toxin Cytotoxicity

Certain embodiments of the cell-targeting molecules of the presentinvention are cytotoxic because they comprise a catalytically active,Shiga toxin effector polypeptide and regardless of the presence of animmunogenic, CD8+ T-cell epitope in the molecule. For example, CD8+T-cell hyper-immunized, Shiga toxin effector polypeptides of the presentinvention, with or without endogenous epitope de-immunization, may beused as components of cell-targeting molecules for applicationsinvolving direct cell-killing, such as, e.g., via the ribotoxic,enzymatic activity of a Shiga toxin effector polypeptide or ribosomebinding and interference with ribosome function due to a non-catalyticmechanism(s).

For certain embodiments of the CD8+ T-cell hyper-immunized,cell-targeting molecules of the present invention, upon contacting acell physically coupled with an extracellular target biomolecule of thecell-targeting binding region, the cell-targeting molecule of theinvention is capable of directly causing the death of the cell, such as,e.g., without the involvement of an untargeted, cytotoxic T-cell (seeSection V-D, supra).

G. Selective Cytotoxicity Among Cell Types

Certain cell-targeting molecules of the present invention have uses inthe selective killing of specific target cells in the presence ofuntargeted, bystander cells. By targeting the delivery of Shiga toxineffector polypeptides of the present invention to specific cells via acell-targeting binding region(s), the cell-targeting molecules of thepresent invention can exhibit cell-type specific, restricted cell-killactivities resulting in the exclusive or preferential killing selectedcell types in the presence of untargeted cells. Similarly, by targetingthe delivery of immunogenic T-cell epitopes to the MHC class I pathwayof target cells, the subsequent presentation of T-cell epitopes andCTL-mediated cytolysis of target cells induced by the cell-targetingmolecules of the invention can be restricted to exclusively orpreferentially killing selected cell types in the presence of untargetedcells. In addition, both the cell-targeted delivery of a cytotoxic,Shiga toxin effector polypeptide region and an immunogenic, T-cellepitope can be accomplished by a single cell-targeting molecule of thepresent invention such that deliver of both potentially cytotoxiccomponents is restricted exclusively or preferentially to target cellsin the presence of untargeted cells.

For certain embodiments, the cell-targeting molecule of the presentinvention is cytotoxic at certain concentrations. In certainembodiments, upon administration of the cell-targeting molecule of thepresent invention to a mixture of cell types, the cytotoxiccell-targeting molecule is capable of selectively killing those cellswhich are physically coupled with an extracellular target biomoleculecompared to cell types not physically coupled with an extracellulartarget biomolecule. For certain embodiments, the cytotoxiccell-targeting molecule of the present invention is capable ofselectively or preferentially causing the death of a specific cell typewithin a mixture of two or more different cell types. This enablestargeting cytotoxic activity to specific cell types with a highpreferentiality, such as a 3-fold cytotoxic effect, over “bystander”cell types that do not express the target biomolecule. Alternatively,the expression of the target biomolecule of the binding region may benon-exclusive to one cell type if the target biomolecule is expressed inlow enough amounts and/or physically coupled in low amounts with celltypes that are not to be targeted. This enables the targetedcell-killing of specific cell types with a high preferentiality, such asa 3-fold cytotoxic effect, over “bystander” cell types that do notexpress significant amounts of the target biomolecule or are notphysically coupled to significant amounts of the target biomolecule.

For certain further embodiments, upon administration of the cytotoxiccell-targeting molecule to two different populations of cell types, thecytotoxic cell-targeting molecule is capable of causing cell death asdefined by the half-maximal cytotoxic concentration (CD₅₀) on apopulation of target cells, whose members express an extracellulartarget biomolecule of the binding region of the cytotoxic cell-targetingmolecule, at a dose at least three-times lower than the CD₅₀ dose of thesame cytotoxic cell-targeting molecule to a population of cells whosemembers do not express an extracellular target biomolecule of thebinding region of the cytotoxic cell-targeting molecule.

For certain embodiments, the cytotoxic activity of a cell-targetingmolecule of the present invention toward populations of cell typesphysically coupled with an extracellular target biomolecule is at least3-fold higher than the cytotoxic activity toward populations of celltypes not physically coupled with any extracellular target biomoleculeof the binding region. According to the present invention, selectivecytotoxicity may be quantified in terms of the ratio (a/b) of (a)cytotoxicity towards a population of cells of a specific cell typephysically coupled with a target biomolecule of the binding region to(b) cytotoxicity towards a population of cells of a cell type notphysically coupled with a target biomolecule of the binding region. Incertain embodiments, the cytotoxicity ratio is indicative of selectivecytotoxicity which is at least 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold,250-fold, 500-fold, 750-fold, or 1000-fold higher for populations ofcells or cell types physically coupled with a target biomolecule of thebinding region compared to populations of cells or cell types notphysically coupled with a target biomolecule of the binding region.

For certain embodiments, the preferential cell-killing function orselective cytotoxicity of a cell-targeting molecule of the presentinvention is due to an additional exogenous material (e.g. a cytotoxicmaterial) and/or heterologous, T-cell epitope present in a Shiga toxineffector polypeptide of the present invention and not necessarily aresult of the catalytic activity of a Shiga toxin effector polypeptideregion.

This preferential cell-killing function allows a targeted cell to bekilled by certain cytotoxic, cell-targeting molecules of the presentinvention under varied conditions and in the presence of non-targetedbystander cells, such as ex vivo manipulated mixtures of cell types, invitro cultured tissues with mixtures of cell types, or in vivo in thepresence of multiple cell types (e.g. in situ or in a native locationwithin a multicellular organism).

H. Delivery of Additional Exogenous Material Into the Interior ofTargeted Cells

In addition to cytotoxic, cytostatic, and immune stimulationapplications, cell-targeting molecules of the present inventionoptionally may be used for targeted intracellular delivery functions,such as, e.g., in applications involving information gathering anddiagnostic functions.

Because the cell-targeting molecules of the invention, including reducedcytotoxicity and/or nontoxic forms thereof, are capable of enteringcells physically coupled with an extracellular target biomoleculerecognized by the cell-targeting molecule’s binding region, certainembodiments of the cell-targeting molecules of the invention may be usedto deliver additional exogenous materials into the interior of targetedcell types. For example, non-toxic variants of the cytotoxic,cell-targeting molecules of the invention, or optionally cytotoxicvariants, may be used to deliver additional exogenous materials toand/or label the interiors of cells physically coupled with anextracellular target biomolecule of the binding region of thecell-targeting molecule. Various types of cells and/or cell populationswhich express target biomolecules to at least one cellular surface maybe targeted by the cell-targeting molecules of the invention forreceiving exogenous materials. The functional components of the presentinvention are modular, in that various Shiga toxin effectorpolypeptides, additional exogenous materials, and binding regions may beassociated with each other to provide cell-targeting molecules suitablefor diverse applications involving cargo delivery, such as, e.g.,non-invasive, in vivo imaging of tumor cells.

This delivery of exogenous material function of certain cell-targetingmolecules of the present invention may be accomplished under variedconditions and in the presence of non-targeted bystander cells, such as,e.g., an ex vivo manipulated target cell, a target cell cultured invitro, a target cell within a tissue sample cultured in vitro, or atarget cell in an in vivo setting like within a multicellular organism.Furthermore, the selective delivery of exogenous material to certaincells by certain cell-targeting molecules of the present invention maybe accomplished under varied conditions and in the presence ofnon-targeted bystander cells, such as ex vivo manipulated mixtures ofcell types, in vitro cultured tissues with mixtures of cell types, or invivo in the presence of multiple cell types (e.g. in situ or in a nativelocation within a multicellular organism).

Shiga toxin effector polypeptides and cell-targeting molecules which arenot capable, such as a certain concentration ranges, of killing a targetcell and/or delivering an embedded or inserted epitope for cell-surfacepresentation by a MHC molecule of a target cell may still be useful fordelivering exogenous materials into cells, such as, e.g., detectionpromoting agents.

For certain embodiments, the Shiga toxin effector polypeptides of thepresent invention exhibits low to zero cytotoxicity and thus arereferred to herein as “non-cytotoxic and/or reduced cytotoxic.” Forcertain embodiments, the cell-targeting molecule of the presentinvention exhibits low to zero cytotoxicity and may be referred to as“non-cytotoxic” and/or “reduced cytotoxic variants.” For example,certain embodiments of the molecules of the present invention do notexhibit a significant level of Shiga toxin based cytotoxicity wherein atdoses of less than 1000 nM, 500 nM, 100 nM, 75 nM, 50 nM, there is nosignificant amount of cell death as compared to the appropriatereference molecule, such as, e.g., as measured by an assay known to theskilled worker and/or described herein. For certain further embodiments,the molecules of the present invention do not exhibit any toxicity atdosages of 1-100 µg per kg of a mammalian recipient. Reduced-cytotoxicvariants may still be cytotoxic at certain concentrations or dosages butexhibit reduced cytotoxicity, such as, e.g., are not capable ofexhibiting a significant level of Shiga toxin cytotoxicity in certainsituations.

Shiga toxin effector polypeptides of the present invention, and certaincell-targeting molecules comprising the same, can be renderednon-cytotoxic, such as, e.g., via the addition of one or more amino acidsubstitutions known to the skilled worker to inactive a Shiga toxin ASubunit and/or Shiga toxin effector polypeptide, including exemplarysubstitutions described herein. The non-cytotoxic and reduced cytotoxicvariants of the cell-targeting molecules of the present invention may bein certain situations more suitable for delivery of additional exogenousmaterials than more cytotoxic variants.

Information Gathering for Diagnostic Functions

In certain cell-targeting molecules of the present invention have usesin the in vitro and/or in vivo detection of specific cells, cell types,and/or cell populations, as well as specific subcellular compartments ofany of the aforementioned. Reduced-cytotoxicity and/or nontoxic forms ofthe cytotoxic, cell-targeting molecules of the invention that areconjugated to detection promoting agents optionally may be used fordiagnostic functions, such as for companion diagnostics used inconjunction with a therapeutic regimen comprising the same or a relatedbinding region, such as, e.g., a binding region with high-affinitybinding to the same target biomolecule, an overlapping epitope, and/orthe same epitope.

In certain embodiments, the cell-targeting molecules described hereinare used for both diagnosis and treatment, or for diagnosis alone. Whenthe same cytotoxic cell-targeting molecule is used for both diagnosisand treatment, for certain embodiments of the present invention thecell-targeting molecule variant which incorporates a detection promotingagent for diagnosis may have its cytotoxicity reduced or may be renderednontoxic by catalytic inactivation of its Shiga toxin effectorpolypeptide region(s) via one or more amino acid substitutions,including exemplary substitutions described herein. For example, certainnontoxic variants of the cell-targeting molecules of the presentinvention exhibit less than 5%, 4%, 3%, 2%, or 1% death of target cellsafter administration of a dose less than 1 mg/kg. Reduced-cytotoxicityvariants may still be cytotoxic at certain concentrations or dosages butexhibit reduced cytotoxicity, such as, e.g., are not capable ofexhibiting a significant level of Shiga toxin cytotoxicity as describedherein.

The ability to conjugate detection promoting agents known in the art tovarious cell-targeting molecules of the present invention providesuseful compositions for the detection of certain cells, such as, e.g.,cancer, tumor, immune, and/or infected cells. These diagnosticembodiments of the cell-targeting molecules of the invention may be usedfor information gathering via various imaging techniques and assaysknown in the art. For example, diagnostic embodiments of thecell-targeting molecules of the invention may be used for informationgathering via imaging of intracellular organelles (e.g. endocytic,Golgi, endoplasmic reticulum, and cytosolic compartments) of individualcancer cells, immune cells, and/or infected cells in a patient or biopsysample.

Various types of information may be gathered using the diagnosticembodiments of the cell-targeting molecules of the invention whether fordiagnostic uses or other uses. This information may be useful, forexample, in diagnosing neoplastic cell types, determining therapeuticsusceptibilities of a patient’s disease, assaying the progression ofanti-neoplastic therapies over time, assaying the progression ofimmunomodulatory therapies over time, assaying the progression ofantimicrobial therapies over time, evaluating the presence of infectedcells in transplantation materials, evaluating the presence of unwantedcell types in transplantation materials, and/or evaluating the presenceof residual tumor cells after surgical excision of a tumor mass.

For example, subpopulations of patients might be ascertained usinginformation gathered using the diagnostic variants of the cell-targetingmolecules of the invention, and then individual patients could befurther categorized into subpopulations based on their uniquecharacteristic(s) revealed using those diagnostic embodiments. Forexample, the effectiveness of specific pharmaceuticals or therapiesmight be a criterion used to define a patient subpopulation. Forexample, a nontoxic diagnostic variant of a particular cytotoxic,cell-targeting molecule of the invention may be used to differentiatewhich patients are in a class or subpopulation of patients predicted torespond positively to a cytotoxic variant of that cell-targetingmolecule of the invention. Accordingly, associated methods for patientidentification, patient stratification, and diagnosis usingcell-targeting molecules of the present invention, including non-toxicvariants of cytotoxic, cell-targeting molecules of the presentinvention, are considered to be within the scope of the presentinvention.

The expression of the target biomolecule by a cell need not be native inorder for cell-targeting by a cell-targeting molecule of the presentinvention, such as, e.g., for direct cell-kill, indirect cell-kill,delivery of exogenous materials like T-cell epitopes, and/or informationgathering. Cell surface expression of the target biomolecule could bethe result of an infection, the presence of a pathogen, and/or thepresence of an intracellular microbial pathogen. Expression of a targetbiomolecule could be artificial such as, for example, by forced orinduced expression after infection with a viral expression vector, seee.g. adenoviral, adeno-associated viral, and retroviral systems.Expression of HER2 can be induced by exposing a cell to ionizingradiation (Wattenberg M et al., Br J Cancer 110: 1472-80 (2014)).

VI. Production, Manufacture, and Purification of Shiga Toxin EffectorPolypeptides of the Invention and Cell-Targeting Molecules Comprisingthe Same

The Shiga toxin effector polypeptides and certain cell-targetingmolecules of the present invention may be produced using techniques wellknown to those of skill in the art. For example, Shiga toxin effectorpolypeptides and cell-targeting molecules of the invention may bemanufactured by standard synthetic methods, by use of recombinantexpression systems, or by any other suitable method. Thus, Shiga toxineffector polypeptides and cell-targeting molecules of the invention maybe synthesized in a number of ways, including, e.g. methods comprising:(1) synthesizing a polypeptide or polypeptide component of acell-targeting molecule using standard solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolating andpurifying the final polypeptide compound product; (2) expressing apolynucleotide that encodes a protein or protein component of acell-targeting molecule of the invention in a host cell and recoveringthe expression product from the host cell or host cell culture; or (3)cell-free, in vitro expression of a polynucleotide encoding apolypeptide or polypeptide component of a cell-targeting molecule of theinvention, and recovering the expression product; or by any combinationof the methods of (1), (2) or (3) to obtain fragments of the proteincomponent, subsequently joining (e.g. ligating) the peptide orpolypeptide fragments to obtain a polypeptide component, and recoveringthe polypeptide component.

It may be preferable to synthesize a Shiga toxin effector polypeptide ofthe present invention, cell-targeting molecule of the present invention,or a protein component of a cell-targeting molecule of the invention bymeans of solid-phase or liquid-phase peptide synthesis. Polypeptides andcell-targeting molecules of the present invention may suitably bemanufactured by standard synthetic methods. Thus, peptides may besynthesized by, e.g. methods comprising synthesizing the peptide bystandard solid-phase or liquid-phase methodology, either stepwise or byfragment assembly, and isolating and purifying the final peptideproduct. In this context, reference may be made to WO 1998/011125 or,inter alia, Fields G et al., Principles and Practice of Solid-PhasePeptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford UniversityPress, U.K., 2nd ed., 2002) and the synthesis examples therein.

Shiga toxin effector polypeptides and cell-targeting molecules of thepresent invention may be prepared (produced and purified) usingrecombinant techniques well known in the art. In general, methods forpreparing proteins by culturing host cells transformed or transfectedwith a vector comprising the encoding polynucleotide and purifying orrecovering the protein from cell culture are described in, e.g.,Sambrook J et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, NY, U.S., 1989); Dieffenbach C et al., PCRPrimer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, N.Y.,U.S., 1995). Any suitable host cell may be used to produce a polypeptideand/or cell-targeting protein of the invention. Host cells may be cellsstably or transiently transfected, transformed, transduced or infectedwith one or more expression vectors which drive expression of apolypeptide of the invention. In addition, a Shiga toxin effectorpolypeptide and/or cell-targeting molecule of the invention may beproduced by modifying the polynucleotide encoding a polypeptide orcell-targeting protein of the invention that result in altering one ormore amino acids or deleting or inserting one or more amino acids inorder to achieve desired properties, such as changed cytotoxicity,changed cytostatic effects, and/or changed serum half-life.

There are a wide variety of expression systems which may be chosen toproduce a polypeptide or cell-targeting protein of the presentinvention. For example, host organisms for expression of cell-targetingproteins of the invention include prokaryotes, such as E. coli and B.subtilis, eukaryotic cells, such as yeast and filamentous fungi (like S.cerevisiae, P. pastoris, A. awamori, and K. lactis), algae (like C.reinhardtii), insect cell lines, mammalian cells (like CHO cells), plantcell lines, and eukaryotic organisms such as transgenic plants (like A.thaliana and N. benthamiana).

Accordingly, the present invention also provides methods for producing aShiga toxin effector polypeptide and/or cell-targeting molecule of thepresent invention according to above recited methods and using apolynucleotide encoding part or all of a polypeptide of the invention ora protein component of a cell-targeting protein of the invention, anexpression vector comprising at least one polynucleotide of theinvention capable of encoding part or all of a polypeptide orcell-targeting protein of the invention when introduced into a hostcell, and/or a host cell comprising a polynucleotide or expressionvector of the invention.

When a protein is expressed using recombinant techniques in a host cellor cell-free system, it is advantageous to separate (or purify) thedesired protein away from other components, such as host cell factors,in order to obtain preparations that are of high purity or aresubstantially homogeneous. Purification can be accomplished by methodswell known in the art, such as centrifugation techniques, extractiontechniques, chromatographic and fractionation techniques (e.g. sizeseparation by gel filtration, charge separation by ion-exchange column,hydrophobic interaction chromatography, reverse phase chromatography,chromatography on silica or cation-exchange resins such as DEAE and thelike, chromatofocusing, and Protein A Sepharose chromatography to removecontaminants), and precipitation techniques (e.g. ethanol precipitationor ammonium sulfate precipitation). Any number of biochemicalpurification techniques may be used to increase the purity of apolypeptide and/or cell-targeting molecule of the present invention. Incertain embodiments, the polypeptides and cell-targeting molecules ofthe invention may optionally be purified in homo-multimeric forms (e.g.a molecular complex comprising two or more polypeptides orcell-targeting molecules of the invention).

In the Examples below are descriptions of non-limiting examples ofmethods for producing exemplary, Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, as well as specificbut non-limiting aspects of production methods.

VII. Pharmaceutical and Diagnostic Compositions ComprisingCell-Targeting Molecules of the Present Invention

The present invention provides Shiga toxin effector polypeptides andcell-targeting molecules for use, alone or in combination with one ormore additional therapeutic agents, in a pharmaceutical composition, fortreatment or prophylaxis of conditions, diseases, disorders, or symptomsdescribed in further detail below (e.g. cancers, malignant tumors,non-malignant tumors, growth abnormalities, immune disorders, andmicrobial infections). For certain embodiments, the one or moreadditional therapeutic agents comprises one or more additionalHER2-targeting therapeutic agent, as described herein. The additionalHER2-targeting therapeutic agent may comprise, consist essentially of,or consist of an anti-HER2 antibody therapy or small molecule inhibitorthat interferes with HER2 signaling. The additional HER2-targetingtherapeutic agent may comprise, consist essentially of, or consist of asmall molecule inhibitor that interferes with HER2 signaling. Forexample, the additional HER2-targeting therapeutic agent may comprise,consist essentially of, or consists of a dual tyrosine kinase inhibitor,such as lapatinib and/or neratinib. For example, the additionalHER2-targeting therapeutic agent may comprise, consist essentially of,or consist of lapatinib. For example, the additional HER2-targetingtherapeutic agent may comprise, consist essentially of, or consist ofneratinib. The additional HER2-targeting therapeutic agent may comprise,consist essentially of, or consist of an anti-HER2 antibody therapy thatbinds to an antigenic determinant that does not overlap with theantigenic determinant bound by the cell-targeting molecule of theinvention or that binds a HER2 molecule in such a manner that when boundthe additional HER2-tageting therapeutic does not prevent the binding ofthat HER2 molecule by the cell-targeting molecule of the invention. Forexample, the additional HER2-targeting therapeutic agent may comprise,consist essentially of, or consist of anti-HER2 monoclonal antibodytherapy and/or anti-HER2 antibody drug conjugate therapy. For example,the additional HER2-targeting therapeutic agent may comprise, consistessentially of, or consist of: T-DM1, trastuzumab, and/ or pertuzumab.For example, the additional HER2-targeting therapeutic agent maycomprise, consist essentially of, or consist of: T-DM1. For example, theadditional HER2-targeting therapeutic agent may comprise, consistessentially of, or consist of: trastuzumab, and/ or pertuzumab. Forexample, the additional HER2-targeting therapeutic agent may comprise,consist essentially of, or consist of trastuzumab. For example, theadditional HER2-targeting therapeutic agent may comprise, consistessentially of, or consist of pertuzumab.

In certain embodiments, the pharmaceutical composition further comprisesat least one pharmaceutically acceptable carrier, excipient, or vehicle,as described herein. The present invention further providespharmaceutical compositions comprising a Shiga toxin polypeptide orcell-targeting molecule of the present invention, or a pharmaceuticallyacceptable salt or solvate thereof, according to the invention, togetherwith at least one pharmaceutically acceptable carrier, excipient, orvehicle. In certain embodiments, the pharmaceutically acceptableexcipient includes a solvent, a dispersion medium, a coating, anantimicrobial agent, an isotonic agent, or an absorption delaying agent;and/or wherein the pharmaceutical composition further comprises anaqueous or non-aqueous carrier; a surfactant; a stabilizer, apreservative, a buffer, an antioxidant, a wetting agent, an emulsifyingagent, a dispersing agent; an isotonic agent; and/or an antibacterial orantifungal agent.

In certain embodiments, the pharmaceutical composition of the presentinvention may comprise homo-multimeric and/or hetero-multimeric forms ofa Shiga toxin effector polypeptides or cell-targeting molecule of thepresent invention. In certain embodiments, the pharmaceuticalcomposition of the present invention may comprise monomeric and/ormonovalent forms of the cell-targeting molecule of the presentinvention. In certain embodiments, the pharmaceutical composition of thepresent invention may be enriched for monomeric and/or monovalent formsof a cell-targeting molecule of the present invention. As demonstratedby the Examples of the application, compositions comprisingpredominantly monovalent and/or monomeric forms of certaincell-targeting molecules may exhibit low levels of toxicity when used invivo while still exhibiting potent cytotoxic to HER2-expressing cells.The pharmaceutical compositions of the invention are useful in methodsof treating, ameliorating, or preventing a disease, condition, disorder,or symptom described in further detail below. The disease, disorder, orcondition may be characterized by cells that are physically coupled withHER2. The HER2 target biomolecule can be physically coupled to thesurface of the cells. In certain embodiments, the disease, disorder orcondition may be characterized by cells that express the HER2 targetbiomolecule (including cells that overexpress HER2). The HER2 can beexpressed (including overexpressed) at the surface of the cells. Eachsuch disease, condition, disorder, or symptom is envisioned to be aseparate embodiment with respect to uses of a pharmaceutical compositionaccording to the invention. The invention further providespharmaceutical compositions for use in at least one method of treatmentaccording to the invention, as described in more detail below.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to any organism, commonly vertebrates such ashumans and animals, which presents symptoms, signs, and/or indicationsof at least one disease, disorder, or condition. These terms includemammals such as the non-limiting examples of primates, livestock animals(e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g.cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats,etc.).

As used herein, “treat,” “treating,” or “treatment” and grammaticalvariants thereof refer to an approach for obtaining beneficial ordesired clinical results. The terms may refer to slowing the onset orrate of development of a condition, disorder or disease, reducing oralleviating symptoms associated with it, generating a complete orpartial regression of the condition, or some combination of any of theabove. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, reduction oralleviation of symptoms, diminishment of extent of disease,stabilization (e.g. not worsening) of state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treat,” “treating,” or “treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterms “treat,” “treating,” or “treatment” includes inhibition orreduction of an increase in severity of a pathological state or symptomsrelative to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant disease, disorder, orcondition. With regard to tumors and/or cancers, treatment includesreduction in overall tumor burden and/or individual tumor size.

As used herein, the terms “prevent,” “preventing,” “prevention” andgrammatical variants thereof refer to an approach for preventing thedevelopment of, or altering the pathology of, a condition, disease, ordisorder. Accordingly, “prevention” may refer to prophylactic orpreventive measures. For the purposes of this invention, beneficial ordesired clinical results include, but are not limited to, prevention orslowing of symptoms, progression or development of a disease, whetherdetectable or undetectable. A subject (e.g. a human) in need ofprevention may thus be a subject not yet afflicted with the disease ordisorder in question. The term “prevention” includes slowing the onsetof disease relative to the absence of treatment, and is not necessarilymeant to imply permanent prevention of the relevant disease, disorder orcondition. Thus “preventing” or “prevention” of a condition may incertain contexts refer to reducing the risk of developing the condition,or preventing or delaying the development of symptoms associated withthe condition.

As used herein, an “effective amount” or “therapeutically effectiveamount” is an amount or dose of a composition (e.g. a therapeuticcomposition, compound, or agent) that produces at least one desiredtherapeutic effect in a subject, such as preventing or treating a targetcondition or beneficially alleviating a symptom associated with thecondition. The most desirable therapeutically effective amount is anamount that will produce a desired efficacy of a particular treatmentselected by one of skill in the art for a given subject in need thereof.This amount will vary depending upon a variety of factors understood bythe skilled worker, including but not limited to the characteristics ofthe therapeutic composition (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type, disease stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject’s response toadministration of a composition and adjusting the dosage accordingly(see e.g. Remington: The Science and Practice of Pharmacy (Gennaro A,ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)).

Diagnostic compositions of the present invention comprise acell-targeting molecule of the present invention and one or moredetection promoting agents. When producing or manufacturing a diagnosticcomposition of the present invention, a cell-targeting molecule of thepresent invention may be directly or indirectly linked to one or moredetection promoting agents. There are numerous standard techniques knownto the skilled worker for incorporating, affixing, and/or conjugatingvarious detection promoting agents to proteins or proteinaceouscomponents of molecules, especially to immunoglobulins andimmunoglobulin-derived domains.

There are numerous detection promoting agents known to the skilledworker, such as isotopes, dyes, colorimetric agents, contrast enhancingagents, fluorescent agents, bioluminescent agents, and magnetic agents,which can be operably linked to the polypeptides or cell-targetingmolecules of the invention for information gathering methods, such asfor diagnostic and/or prognostic applications to diseases, disorders, orconditions of an organism (see e.g. Cai W et al., J Nucl Med 48: 304-10(2007); Nayak T, Brechbiel M, Bioconjug Chem 20: 825-41 (2009); PaudyalP et al., Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6: e18103(2011); Sano K et al., Breast Cancer Res 14: R61 (2012)). These agentsmay be associated with, linked to, and/or incorporated within thepolypeptide or cell-targeting molecule of the invention at any suitableposition. For example, the linkage or incorporation of the detectionpromoting agent may be via an amino acid residue(s) of a molecule of thepresent invention or via some type of linkage known in the art,including via linkers and/or chelators. The incorporation of the agentis in such a way to enable the detection of the presence of thediagnostic composition in a screen, assay, diagnostic procedure, and/orimaging technique.

Similarly, there are numerous imaging approaches known to the skilledworker, such as non-invasive in vivo imaging techniques commonly used inthe medical arena, for example: computed tomography imaging (CTscanning), optical imaging (including direct, fluorescent, andbioluminescent imaging), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), ultrasound, and x-ray computed tomography imaging.

VIII. Production or Manufacture of Pharmaceutical and/or DiagnosticCompositions Comprising Cell-Targeting Molecules of the PresentInvention

Pharmaceutically acceptable salts or solvates of any of the Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention are within the scope of the present invention.

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, aproteinaceous compound or pharmaceutically acceptable salt thereofaccording to the invention) and a solvent. The solvent in thisconnection may, for example, be water, ethanol or anotherpharmaceutically acceptable, typically small-molecular organic species,such as, but not limited to, acetic acid or lactic acid. When thesolvent in question is water, such a solvate is normally referred to asa hydrate.

Polypeptides and proteins of the present invention, or salts thereof,may be formulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a molecule of the present invention, or a salt thereof, in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” includes any of the standard pharmaceuticalcarriers. Pharmaceutically acceptable carriers for therapeutic moleculeuse are well known in the pharmaceutical art, and are described, forexample, in Remington’s Pharmaceutical Sciences (Mack Publishing Co. (A.Gennaro, ed., 1985). As used herein, “pharmaceutically acceptablecarrier” includes any and all physiologically acceptable, i.e.compatible, solvents, dispersion media, coatings, antimicrobial agents,isotonic, and absorption delaying agents, and the like. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. Exemplary pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers thatmay be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. In certain embodiments, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onselected route of administration, the protein or other pharmaceuticalcomponent may be coated in a material intended to protect the compoundfrom the action of low pH and other natural inactivating conditions towhich the active protein may encounter when administered to a patient bya particular route of administration.

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for therapeuticproteins described herein.

The pharmaceutical compositions of the present invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Preventing the presence of microorganismsmay be ensured both by sterilization procedures, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, suchas sugars, sodium chloride, and the like into the compositions, may alsobe desirable. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as, aluminum monostearate and gelatin.

A pharmaceutical composition of the present invention also optionallyincludes a pharmaceutically acceptable antioxidant. Exemplarypharmaceutically acceptable antioxidants are water soluble antioxidantssuch as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and thelike; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, andthe like.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different polypeptidesand/or cell-targeting molecules of the invention, or an ester, salt oramide of any of the foregoing, and at least one pharmaceuticallyacceptable carrier.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g., sugars andpolyalcohols such as mannitol, sorbitol, or sodium chloride, may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating apolypeptide or cell-targeting molecule of the invention in the requiredamount in an appropriate solvent with one or a combination ofingredients described above, as required, followed by sterilizationmicrofiltration. Dispersions may be prepared by incorporating the activecompound into a sterile vehicle that contains a dispersion medium andother ingredients, such as those described above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient in addition to any additionaldesired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a polypeptide and/orcell-targeting molecule of the invention is designed to be administeredby, e.g. intravenous, cutaneous or subcutaneous injection, the bindingagent will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. Methods for preparing parenterally acceptable proteinsolutions, taking into consideration appropriate pH, isotonicity,stability, and the like, are within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection will contain, in addition to binding agents, an isotonicvehicle such as sodium chloride injection, Ringer’s injection, dextroseinjection, dextrose and sodium chloride injection, lactated Ringer’sinjection, or other vehicle as known in the art. A pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives well known tothose of skill in the art.

As described elsewhere herein, a polypeptide and/or cell-targetingmolecule of the present invention may be prepared with carriers thatwill protect the active therapeutic agent against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art (see e.g.Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed.,Marcel Dekker, Inc., NY, U.S., 1978)).

In certain embodiments, the pharmaceutical composition of the presentinvention comprises a buffer, such as e.g., citrate, citric acid,histidine, phosphate, succinate, and/or succinic acid. In certainembodiments, the pharmaceutical composition of the present inventioncomprises a preservative, antibacterial, or antifungal agent, such ase.g., mannitol or sorbitol. In certain embodiments, the pharmaceuticalcomposition of the present invention comprises a detergent such as,e.g., polysorbate 20 or polysorbate 80. In certain embodiments, thepharmaceutical composition of the present invention comprises acryoprotectant such as, e.g., polysorbate 20 or polysorbate 80. Incertain embodiments, the pharmaceutical composition of the presentinvention comprises an excipient, such as, e.g., arginine, argininesulfate, glycerol, mannitol, methionine, polysorbate 20, polysorbate 80,sorbitol, sucrose, and/or trehalose. In certain further embodiments, thepharmaceutical composition of the present invention comprises one ormore of (including all of): citrate, polysorbate 20, sodium, sorbitol,and chloride. In certain further embodiments, the pharmaceuticalcomposition comprises a 20 millimolar concentration of citrate, 200millimolar concentration of sorbitol, and 0.2% polysorbate 20. Incertain further embodiments, at room temperature (e.g. about 25° C.) thepharmaceutical composition has a pH of about 5.3 to 5.7, a pH between5.4 and 5.6, and/or a pH of 5.5.

In certain embodiments, the composition of the present invention (e.g. apharmaceutical and/or diagnostic composition) may be formulated toensure a desired in vivo distribution of a cell-targeting molecule ofthe present invention. For example, the blood-brain barrier excludesmany large and/or hydrophilic compounds. To target a therapeuticmolecule or composition of the present invention to a particular in vivolocation, they can be formulated, for example, in liposomes which maycomprise one or more moieties that are selectively transported intospecific cells or organs, thus enhancing targeted drug delivery.Exemplary targeting moieties include folate or biotin; mannosides;antibodies; surfactant protein A receptor; p120 catenin and the like.

Pharmaceutical compositions include parenteral formulations designed tobe used as implants or particulate systems. Examples of implants aredepot formulations composed of polymeric or hydrophobic components suchas emulsions, ion exchange resins, and soluble salt solutions. Examplesof particulate systems are microspheres, microparticles, nanocapsules,nanospheres, and nanoparticles (see e.g. Honda M et al., Int JNanomedicine 8: 495-503 (2013); Sharma A et al., Biomed Res Int 2013:960821 (2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).Controlled release formulations may be prepared using polymers sensitiveto ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.

The pharmaceutically acceptable carrier in the pharmaceuticalcompositions of the present invention may comprise: a physiologicallyacceptable solvent, dispersion medium, coating, antimicrobial agent,isotonic agent, absorption delaying agent, sterile aqueous solution ordispersion, or sterile powder; an aqueous or non-aqueous carrier, suchas water, alcohol (e.g. ethanol), polyol (e.g. glycerol, propyleneglycol, or polyethylene glycol), and mixtures thereof; vegetable oil; oran injectable organic ester, such as ethyloleate. The pharmaceuticalcomposition of the invention may further comprises an adjuvant, such asa preservative, wetting agent, emulsifying agent, or dispersing agent;an antibacterial or antifungal agent, such as a paraben, chlorobutanol,phenol, or sorbic acid; an isotonic agent, such as a sugar, apolyalcohol such as mannitol or sorbitol, or sodium chloride; anabsorption-delaying agent, such as aluminum monostearate or gelatin; acoating, such as lecithin; a pharmaceutically acceptable antioxidant; asurfactant; a buffer; and/or a stabilizer. In certain embodiments, thepharmaceutically acceptable antioxidant is a water soluble antioxidant,such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, or sodium sulfite; an oil-soluble antioxidant, such asascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propylgallate, or alpha-tocopherol; or ametal chelating agent, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, or phosphoric acid.

IX. Polynucleotides, Expression Vectors, and Host Cells of the PresentInvention

Beyond the polypeptides and cell-targeting molecules of the presentinvention, the polynucleotides that encode the polypeptides andcell-targeting molecules of the invention, or functional portionsthereof, are also encompassed within the scope of the present invention.The term “polynucleotide” is equivalent to the term “nucleic acid,” eachof which includes one or more of: polymers of deoxyribonucleic acids(DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs orRNAs generated using nucleotide analogs, and derivatives, fragments andhomologs thereof. The polynucleotide of the present invention may besingle-, double-, or triple-stranded. Such polynucleotides arespecifically disclosed to include all polynucleotides capable ofencoding an exemplary protein, for example, taking into account thewobble known to be tolerated in the third position of RNA codons, yetencoding for the same amino acid as a different RNA codon (see StothardP, Biotechniques 28: 1102-4 (2000)).

In one aspect, the present invention provides polynucleotides whichencode a Shiga toxin effector polypeptide and/or cell-targeting moleculeof the present invention, or a fragment or derivative thereof. Thepolynucleotides may include, e.g., a nucleic acid sequence encoding apolypeptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or more, identical to a polypeptide comprising one of the amino acidsequences of a polypeptide or cell-targeting molecule of the presentinvention. The invention also includes polynucleotides comprisingnucleotide sequences that hybridize under stringent conditions to apolynucleotide which encodes Shiga toxin effector polypeptide and/orcell-targeting molecule of the invention, or a fragment or derivativethereof, or the antisense or complement of any such sequence.

Derivatives or analogs of the molecules of the present invention (e.g.,Shiga toxin effector polypeptides of the present invention andcell-targeting molecules comprising the same) include, inter alia,polynucleotide (or polypeptide) molecules having regions that aresubstantially homologous to the polynucleotides (or Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention),e.g. by at least about 45%, 50%, 70%, 80%, 85%, 90%, 95%, 98%, or even99% identity (with a preferred identity of 80-99%) over a polynucleotide(or polypeptide) sequence of the same size or when compared to analigned sequence in which the alignment is done by a computer homologyprogram known in the art. An exemplary program is the GAP program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, WI, U.S.) using thedefault settings, which uses the algorithm of Smith T, Waterman M, AdvAppl Math 2: 482-9 (1981). Also included are polynucleotides capable ofhybridizing to the complement of a sequence encoding the cell-targetingproteins of the invention under stringent conditions (see e.g. Ausubel Fet al., Current Protocols in Molecular Biology (John Wiley & Sons, NewYork, NY, U.S., 1993)), and below. Stringent conditions are known tothose skilled in the art and may be found, e.g., in Current Protocols inMolecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6(1989)).

The present invention further provides expression vectors that comprisethe polynucleotides within the scope of the present invention. Thepolynucleotides capable of encoding the Shiga toxin effectorpolypeptides and/or cell-targeting molecules of the invention may beinserted into known vectors, including bacterial plasmids, viral vectorsand phage vectors, using material and methods well known in the art toproduce expression vectors. Such expression vectors will include thepolynucleotides necessary to support production of contemplated Shigatoxin effector polypeptides and/or cell-targeting molecules of theinvention within any host cell of choice or cell-free expression systems(e.g. pTxb1 and pIVEX2.3). The specific polynucleotides comprisingexpression vectors for use with specific types of host cells orcell-free expression systems are well known to one of ordinary skill inthe art, can be determined using routine experimentation, and/or may bepurchased.

The term “expression vector,” as used herein, refers to apolynucleotide, linear or circular, comprising one or more expressionunits. The term “expression unit” denotes a polynucleotide segmentencoding a polypeptide of interest and capable of providing expressionof the nucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. An expression vector contains one or more expressionunits. Thus, in the context of the present invention, an expressionvector encoding a Shiga toxin effector polypeptide and/or cell-targetingmolecule of the invention comprising a single polypeptide chain includesat least an expression unit for the single polypeptide chain, whereas aprotein comprising, e.g. two or more polypeptide chains (e.g. one chaincomprising a V_(L) domain and a second chain comprising a V_(H) domainlinked to a toxin effector polypeptide) includes at least two expressionunits, one for each of the two polypeptide chains of the protein. Forexpression of multi-chain cell-targeting proteins of the invention, anexpression unit for each polypeptide chain may also be separatelycontained on different expression vectors (e.g. expression may beachieved with a single host cell into which expression vectors for eachpolypeptide chain has been introduced).

Expression vectors capable of directing transient or stable expressionof polypeptides and proteins are well known in the art. The expressionvectors generally include, but are not limited to, one or more of thefollowing: a heterologous signal sequence or peptide, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, each of which is well known inthe art. Optional regulatory control sequences, integration sequences,and useful markers that can be employed are known in the art.

The term “host cell” refers to a cell which can support the replicationor expression of the expression vector. Host cells may be prokaryoticcells, such as E. coli or eukaryotic cells (e.g. yeast, insect,amphibian, bird, or mammalian cells). Creation and isolation of hostcell lines comprising a polynucleotide of the invention or capable ofproducing a polypeptide and/or cell-targeting molecule of the presentinvention can be accomplished using standard techniques known in theart.

Shiga toxin effector polypeptides and/or proteins within the scope ofthe present invention may be variants or derivatives of the polypeptidesand molecules described herein that are produced by modifying thepolynucleotide encoding a polypeptide and/or proteinaceous component ofa cell-targeting molecule by altering one or more amino acids ordeleting or inserting one or more amino acids that may render it moresuitable to achieve desired properties, such as more optimal expressionby a host cell.

X. Molecules of the Present Invention Immobilized on Solid Substrates

Certain embodiments of the present invention include a molecule of thepresent invention (e.g. a Shiga toxin effector polypeptide, acell-targeting molecule, fusion protein, or polynucleotide of thepresent invention), or any effector fragment thereof, immobilized on asolid substrate. Solid substrates contemplated herein include, but arenot limited to, microbeads, nanoparticles, polymers, matrix materials,microarrays, microtiter plates, or any solid surface known in the art(see e.g. US 7,771,955). In accordance with these embodiments, amolecule of the present invention may be covalently or non-covalentlylinked to a solid substrate, such as, e.g., a bead, particle, or plate,using techniques known to the skilled worker (see e.g. Jung Y et al.,Analyst 133: 697-701 (2008)). Immobilized molecules of the presentinvention (e.g. a HER2-targeting molecule which comprises, consists of,or consists essentially of any one of SEQ ID NOs: 29, 36, 102, and 108)may be used for screening applications using techniques known in the art(see e.g. Bradbury A et al., Nat Biotechnol 29: 245-54 (2011); Sutton C,Br J Pharmacol 166: 457-75 (2012); Diamante L et al., Protein Eng DesSel 26: 713-24 (2013); Houlihan G et al., J Immunol Methods 405: 47-56(2014)).

Non-limiting examples of solid substrates to which a molecule of theinvention may be immobilized on include: microbeads, nanoparticles,polymers, nanopolymers, nanotubes, magnetic beads, paramagnetic beads,superparamagnetic beads, streptavidin coated beads, reverse-phasemagnetic beads, carboxy terminated beads, hydrazine terminated beads,silica (sodium silica) beads and iminodiacetic acid (IDA) -modifiedbeads, aldehyde-modified beads, epoxy-activated beads,diaminodipropylamine (DADPA) -modified beads (beads with primary aminesurface group), biodegradable polymeric beads, polystyrene substrates,amino-polystyrene particles, carboxyl-polystyrene particles,epoxy-polystyrene particles, dimethylamino-polystyrene particles,hydroxy-polystyrene particles, colored particles, flow cytometryparticles, sulfonate-polystyrene particles, nitrocellulose surfaces,reinforced nitrocellulose membranes, nylon membranes, glass surfaces,activated glass surfaces, activated quartz surfaces, polyvinylidenedifluoride (PVDF) membranes, polyacrylamide-based substrates, poly-vinylchloride substrates, poly-methyl methacrylate substrates, poly(dimethylsiloxane) substrates, and photopolymers which contain photoreactivespecies (such as nitrenes, carbenes, and ketyl radicals) capable offorming covalent linkages. Other examples of solid substrates to which amolecule of the invention may be immobilized on are commonly used inmolecular display systems, such as, e.g., cellular surfaces, phages, andvirus particles.

XI. Delivery Devices and Kits

In certain embodiments, the invention relates to a device comprising oneor more compositions of matter of the present invention, such as apharmaceutical composition or diagnostic composition, for delivery to asubject in need thereof. Thus, a delivery device comprising one or morecompositions of the present invention can be used to administer to apatient a composition of matter of the present invention by variousdelivery methods, including: intravenous, subcutaneous, intramuscular orintraperitoneal injection; oral administration; transdermaladministration; pulmonary or transmucosal administration; administrationby implant, osmotic pump, cartridge or micro pump; or by other meansrecognized by a person of skill in the art.

Also within the scope of the present invention are kits comprising atleast one composition of matter of the invention, and optionally,packaging and instructions for use. For example, the present inventionprovides a kit comprising: (i) a HER2-targeting molecule of the presentinvention, (ii) a pharmaceutical composition of the present invention,(iii) a diagnostic composition of the present invention, (iv) apolynucleotide of the present invention, (v) an expression vector of thepresent invention and/or (vi) a host cell of the present invention; andoptionally, packaging and instructions for use. Kits may be useful fordrug administration and/or diagnostic information gathering. A kit ofthe invention may optionally comprise at least one additional reagent(e.g., standards, markers and the like). Kits typically include a labelindicating the intended use of the contents of the kit. The kit mayfurther comprise reagents and other tools for detecting a cell type(e.g. a tumor cell) in a sample or in a subject, or for diagnosingwhether a patient belongs to a group that responds to a therapeuticstrategy which makes use of a compound, composition, or related methodof the present invention, e.g., such as a method described herein.

XII. Methods for Using Cell-Targeting Molecules of the Present Inventionand/or Pharmaceutical and/or Diagnostic Compositions Thereof

Generally, it is an object of the present invention to providepharmacologically active agents, as well as compositions comprising thesame, that can be used in the prevention and/or treatment of diseases,disorders, and conditions, such as certain cancers, tumors, growthabnormalities, immune disorders, or further pathological conditionsmentioned herein. Accordingly, the present invention provides methods ofusing the polypeptides, cell-targeting molecules, and pharmaceuticalcompositions of the invention for the targeted killing of cells, fordelivering additional exogenous materials into targeted cells, forlabeling of the interiors of targeted cells, for collecting diagnosticinformation, for the delivering of T-cell epitopes to the MHC class Ipresentation pathway of target cells, and for treating diseases,disorders, and conditions as described herein. For example, the methodsof the present invention may be used to prevent or treat cancers, cancerinitiation, tumor initiation, metastasis, and/or disease reoccurrence.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions, and/or methods that havecertain advantages compared to the agents, compositions, and/or methodsthat are currently known in the art. Accordingly, the present inventionprovides methods of using Shiga toxin effector polypeptides andcell-targeting molecules with specified protein sequences andpharmaceutical compositions thereof. For example, any of the amino acidsequences described herein may be specifically utilized as a componentof the cell-targeting molecule used in the following methods or anymethod for using a cell-targeting molecule known to the skilled worker,such as, e.g., various methods described in WO 2014/164680, WO2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO2015/113007, WO 2015/191764, US20150259428, WO 2016/196344, WO2017/019623, and WO 2018/140427.

The present invention provides methods of killing a cell comprising thestep of contacting the cell, either in vitro or in vivo, with a Shigatoxin effector polypeptide, cell-targeting molecule, or pharmaceuticalcomposition of the present invention. The Shiga toxin effectorpolypeptides, cell-targeting molecules, and pharmaceutical compositionsof the present invention can be used to kill a specific cell type uponcontacting a cell or cells with one of the claimed compositions ofmatter. For certain embodiments, the cell(s) is physically coupled withHER2. For certain embodiments, the cell(s) expresses (includingover-expresses) HER2. The HER2 may be expressed (includingoverexpressed) at the surface of the cells. For certain embodiments, acell-targeting molecule or pharmaceutical composition of the presentinvention can be used to kill specific cell types in a mixture ofdifferent cell types, such as mixtures comprising cancer cells, infectedcells, and/or hematological cells. For certain embodiments, acell-targeting molecule, or pharmaceutical composition of the presentinvention can be used to kill cancer cells in a mixture of differentcell types. For certain embodiments, a cytotoxic Shiga cell-targetingmolecule, or pharmaceutical composition of the present invention can beused to kill specific cell types in a mixture of different cell types,such as pre-transplantation tissues. For certain embodiments, a Shigatoxin effector polypeptide, cell-targeting molecule, or pharmaceuticalcomposition of the present invention can be used to kill specific celltypes in a mixture of cell types, such as pre-administration tissuematerial for therapeutic purposes. For certain embodiments, acell-targeting molecule or pharmaceutical composition of the presentinvention can be used to selectively kill cells infected by viruses ormicroorganisms, or otherwise selectively kill cells expressing aparticular extracellular target biomolecule, such as a cell surfacelocalized HER2 variant. The Shiga toxin effector polypeptides,cell-targeting molecules, and pharmaceutical compositions of the presentinvention have varied applications, including, e.g., uses in depletingunwanted cell types from tissues either in vitro or in vivo, uses asantiviral agents, and uses in purging transplantation tissues ofunwanted cell types. For certain embodiments, the cell expresses muc-4and/or CD44. For certain embodiments, the cell is resistant tocytotoxicity caused by T-DM1 (trastuzumab emtansine) and/or trastuzumab.For certain further embodiments the cell(s) are in the presence ofpertuzumab, T-DM1 (trastuzumab emtansine), lapatinib, and/or neratinib;and/or had previously been contacted with pertuzumab, T-DM1 (trastuzumabemtansine), lapatinib, and/or neratinib. Among certain embodiments ofthe present invention is a method of killing a cell (e.g. aHER2-expressing cell) comprising the step of contacting the cell withthe cell-targeting molecule of the present invention or thepharmaceutical composition of the present invention wherein the cell isin the presence of pertuzumab, T-DM1 (trastuzumab emtansine), lapatinib,and/or neratinib; and/or had previously been contacted with pertuzumab,T-DM1 (trastuzumab emtansine), lapatinib and/or neratinib. For certainfurther embodiments the cell(s) are in the presence of T-DM1(trastuzumab emtansine). For certain further embodiments the cell(s) arein the presence of pertuzumab. For certain further embodiments thecell(s) are in the presence of lapatinib. For certain furtherembodiments the cell(s) are in the presence of neratinib. For certainfurther embodiments the cell(s) had previously been contacted withpertuzumab. For certain further embodiments the cell(s) had previouslybeen contacted with T-DM1 (trastuzumab emtansine). For certain furtherembodiments the cell(s) had previously been contacted with lapatinib.For certain further embodiments the cell(s) had previously beencontacted with neratinib.

For certain embodiments, certain Shiga toxin effector polypeptides,cell-targeting molecules, and pharmaceutical compositions of the presentinvention, alone or in combination with other compounds orpharmaceutical compositions, can show potent cell-kill activity whenadministered to a population of cells, in vitro or in vivo in a subjectsuch as in a patient in need of treatment. By targeting the delivery ofenzymatically active Shiga toxin A Subunit effector polypeptides and/orT-cell epitopes using high-affinity binding regions to specific celltypes, cell-kill activities can be restricted to specifically andselectively killing certain cell types within an organism, such ascertain cancer cells, neoplastic cells, malignant cells, non-malignanttumor cells, and/or infected cells.

The present invention provides a method of killing a cell in a patientin need thereof, the method comprising the step of administering to thepatient at least one cell-targeting molecule of the present invention ora pharmaceutical composition thereof.

For certain embodiments, the cell-targeting molecule of the presentinvention or pharmaceutical compositions thereof can be used to kill acancer cell in a patient by targeting an extracellular biomolecule foundphysically coupled with a cancer or tumor cell. The terms “cancer cell”or “cancerous cell” refers to various neoplastic cells which grow anddivide in an abnormally accelerated and/or unregulated fashion and willbe clear to the skilled person. The term “tumor cell” includes bothmalignant and non-malignant cells. Generally, cancers and/or tumors canbe defined as diseases, disorders, or conditions that are amenable totreatment and/or prevention. The cancers and tumors (either malignant ornon-malignant) which are comprised of cancer cells and/or tumor cellswhich may benefit from methods and compositions of the invention will beclear to the skilled person. Neoplastic cells are often associated withone or more of the following: unregulated growth, lack ofdifferentiation, local tissue invasion, angiogenesis, and metastasis.The diseases, disorders, and conditions resulting from cancers and/ortumors (either malignant or non-malignant) which may benefit from themethods and compositions of the present invention targeting certaincancer cells and/or tumor cells will be clear to the skilled person. Forexample, disease, disorder, or condition may be characterized by cellsthat are physically coupled with HER2. The HER2 target biomolecule maybe physically coupled to the surface of the cells. For certainembodiments, the disease, disorder or condition may be characterized bycells that express the HER2 target biomolecule (including cells thatoverexpress HER2). The HER2 may be expressed (including overexpressed)at the surface of the cells.

Certain embodiments of the cell-targeting molecules and compositions ofthe present invention may be used to treat diseases, disorders orconditions (such as, e.g., HER2 positive cancers and/or tumors) in apatient after the patient has already received a HER2-targetedtherapeutic agent. In many situations, cell-surface HER2 expressionpersists during disease progression after a therapeutic treatment suchas, e.g., a HER2-targeted therapy using an anti-HER2 monoclonal antibodytherapy or anti-HER2 antibody drug conjugate therapy, or achemotherapeutic agent therapy using a tyrosine kinase inhibitor. Thus,HER2 is still present as a target on the surfaces of malignant/targetcells and available for targeting by a cell-targeting molecule of thepresent invention for cell-surface docking and cellular internalization.Furthermore, as demonstrated by the Examples, the cell-targetingmolecules of the present invention (and compositions comprising thecell-targeting molecules) can be used in combination with otherHER2-targeted therapeutic agents, such as the anti-HER2 antibodytherapies that bind to non-overlapping antigenic determinants of HER2;or the tyrosine kinase inhibitors that have a different HER2-targetingactivity to the cell-targeting molecules of the invention. Accordingly,the “patient in need thereof” that is administered with at least onecell-targeting molecule or a pharmaceutical composition thereof in themethods of the present invention, includes a patient(s) that has beenpreviously treated with an additional HER2-targeting therapeutic agent;and/or is undergoing treatment with an additional HER2-targetingtherapeutic agent. For certain embodiments, the patient(s) has beenpreviously treated with an additional HER2-targeting therapeutic agentas described herein. For certain embodiments, the patient(s) isundergoing treatment with an additional HER2-targeting therapeutic agentas described herein. For certain embodiments, the “patient in needthereof” does not respond to, or does not benefit from, treatment withone or more additional HER2-targeting therapeutic agent. For example,this can be due to, inter alia, acquired and/or intrinsic resistance.For certain embodiments, the additional HER2-targeting therapeutic agentcomprises a tyrosine kinase inhibitor, an anti-HER2 monoclonal antibodytherapy or an anti-HER2 antibody drug conjugate therapy. For certainembodiments, the additional HER2-targeting therapeutic agent comprisesone or more of: pertuzumab, trastuzumab, T-DM1 (trastuzumab emtansine),lapatinib and/or neratinib. For certain embodiments, the additionalHER2-targeting therapeutic agent is pertuzumab. For certain embodiments,the additional HER2-targeting therapeutic agent is trastuzumab. Forcertain embodiments, additional HER2-targeting therapeutic agent isT-DM1 (trastuzumab emtansine). For certain embodiments, the additionalHER2-targeting therapeutic agent is lapatinib. For certain embodiments,the additional HER2-targeting therapeutic agent is neratinib.

As used herein, the reference to “a patient in need thereof” that “hasbeen previously treated with an additional HER2-targeting therapeuticagent” includes patients that were last administered treatment with anadditional HER2-targeting therapeutic agent at least 6 months (such asat least 5 months, 4 months, 3 months, 2 months or 1 month), at least 6weeks (such as at least 5 weeks, 4 weeks, 3 weeks, 2 weeks or 1 week) orat least 144 hours (such as at least 120 hours, 96 hours, 72 hours, 48hours, 24 hours, 12 hours, or 6 hours) prior to treatment with thecell-targeting molecule or pharmaceutical composition of the presentinvention.

As used herein, the reference to “a patient in need thereof” that “isundergoing treatment with an additional HER2-targeting therapeuticagent” includes patients that are simultaneously or sequentiallyadministered with the cell-targeting molecule or pharmaceuticalcomposition of the present invention and an additional HER2-targetingtherapeutic agent. The patient may be administered with the additionalHER2-targeting therapeutic agent at least 1 hour (such as at least 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or144 hours), 1 week (such as at least 2 weeks, 3 weeks, 4 weeks, 5 weeksor 6 weeks) or 1 month (such as at least 2 months, 3 months, 4 months, 5months or 6 months) prior to, or subsequent to, treatment with thecell-targeting molecule or pharmaceutical composition of the presentinvention.

As used herein, the reference to a “patient in need thereof” that doesnot respond to, or does not benefit from, treatment with one or moreadditional HER2-targeting therapeutic agent includes patients that areresistant to or have developed resistance to the one or more additionalHER2-targeting therapeutic agent. For examples, drug resistance mayarise from the expression of drug efflux pumps or cytochrome P450enzymes (e.g. CYP3A4) as well as obstacles preventing HER2 epitopebinding, e.g. HER2 epitope masking of the epitope bound by theHER2-targeting therapeutic. For example, drug resistance may arise fromthe existence of activated survival/proliferation pathways redundant toHER2 signaling or downstream of HER2 activity thereby bypassing HER2.For example, drug resistance may arise from the existence of mutationsin HER2 that alter the drug’s effectiveness, such as, e.g., mutations inthe ATP-binding pocket bound by a HER2 inhibitor. Resistance mechanismstied to the additional HER2-targeting therapeutic agent mechanism ofaction can be avoided by HER2-targeting molecules of the presentinvention that effectuating a different mechanism of action.

Certain embodiments of the cell-targeting molecules and compositions ofthe present invention may be used to treat cancers and/or tumors in asubject after the subject has already received a HER2-targeted therapy.In many situations HER2 persists during disease progression after atherapeutic treatment such as, e.g., a HER2-targeted therapy using ananti-HER2 monoclonal antibody therapy or anti-HER2 antibody drugconjugate therapy, or a chemotherapeutic agent therapy using a tyrosinekinase inhibitor. Thus, HER2 is still present as a target on thesurfaces of malignant/target cells and available for targeting by acell-targeting molecule of the present invention.

Certain embodiments of the cell-targeting molecules and compositions ofthe present invention may be used to kill cancer stem cells, tumor stemcells, pre-malignant cancer-initiating cells, and tumor-initiatingcells, which commonly are slow dividing and resistant to cancertherapies like chemotherapy and radiation.

Because of the Shiga toxin A Subunit based mechanism of action,compositions of matter of the present invention may be more effectivelyused in methods involving their combination with, or in complementaryfashion with other therapies, such as, e.g., chemotherapies,immunotherapies, radiation, stem cell transplantation, and immunecheckpoint inhibitors, and/or effective againstchemoresistant/radiation-resistant and/or resting tumor cells/tumorinitiating cells/stem cells. Similarly, compositions of matter of thepresent invention may be more effectively used in methods involving incombination with other cell-targeted therapies targeting other than thesame epitope on, non-overlapping, or different targets for the samedisease disorder or condition. These other therapies or othercell-targeted therapies include the additional HER2-targetingtherapeutic agent(s) described herein.

Certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to killan immune cell (whether healthy or malignant) in a patient by targetingan extracellular biomolecule found physically coupled with an immunecell.

For certain embodiments of the cell-targeting molecule of the presentinvention, or pharmaceutical compositions thereof, can be used to killan infected cell in a patient by targeting an extracellular biomoleculefound physically coupled with an infected cell.

For certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to “seed”a locus within a chordate with non-self, T-cell epitope-peptidepresenting cells in order to activate the immune system to enhancepolicing of the locus. For certain further embodiments of this “seeding”method of the present invention, the locus is a tumor mass or infectedtissue site. In preferred embodiments of this “seeding” method of thepresent invention, the non-self, T-cell epitope-peptide is selected fromthe group consisting of: peptides not already presented by the targetcells of the cell-targeting molecule, peptides not present within anyprotein expressed by the target cell, peptides not present within theproteome or transcriptome of the target cell, peptides not present inthe extracellular microenvironment of the site to be seeded, andpeptides not present in the tumor mass or infect tissue site to betargeting.

This “seeding” method functions to label one or more target cells withina chordate with one or more MHC class I presented T-cell epitopes forrecognition by effector T-cells and activation of downstream immuneresponses. By exploiting the cell internalizing, intracellularlyrouting, and T-cell epitope delivering functions of the cell-targetingmolecules of the present invention, the target cells which display thedelivered T-cell epitope are harnessed to induce recognition of thepresenting target cell by host T-cells and induction of further immuneresponses including target-cell-killing by CTLs. This “seeding” methodof using a cell-targeting molecule of the present invention can providea temporary vaccination-effect by inducing adaptive immune responses toattack the cells within the seeded microenvironment, such as, e.g. atumor mass or infected tissue site, whether presenting a cell-targetingmolecule-delivered T-cell epitope(s) or not. This “seeding” method mayalso induce the breaking of immuno-tolerance to a target cellpopulation, a tumor mass, and/or infected tissue site within a chordate.

Certain methods of the present invention involving the seeding of alocus within a chordate with one or more antigenic and/or immunogenicepitopes may be combined with the administration of immunologicadjuvants, whether administered locally or systemically, to stimulatethe immune response to certain antigens, such as, e.g., theco-administration of a composition of the present invention with one ormore immunologic adjuvants like a cytokine, bacterial product, or plantsaponin. Other examples of immunologic adjuvants which may be suitablefor use in the methods of the present invention include aluminum saltsand oils, such as, e.g., alums, aluminum hydroxide, mineral oils,squalene, paraffin oils, peanut oils, and thimerosal.

Additionally, the present invention provides a method of treating adisease, disorder, or condition in a patient, the method comprising thestep of administering to a patient in need thereof a therapeuticallyeffective amount of at least one of the cell-targeting molecules of thepresent invention, or a pharmaceutical composition thereof. The disease,disorder or condition may be characterized by cells that are physicallycoupled with HER2/neu/ErbB2. The HER2/neu/ErbB2 may be physicallycoupled to the surface of the cells. For certain embodiments, thedisease, disorder or condition may be characterized by cells thatexpress (including overexpress) HER2/neu/ErbB2. The HER2/neu/ErbB2 maybe expressed (including overexpressed) at the surface of the cells.Contemplated diseases, disorders, and conditions that can be treatedusing this method include cancers, malignant tumors, non-malignanttumors, growth abnormalities, immune disorders, and microbialinfections. The cancer, tumor, growth abnormality, immune disorder, ormicrobial infection may be characterized by cells that are physicallycoupled with HER2/neu/ErbB2. The HER2/neu/ErbB2 may be physicallycoupled to the surface of the cells. For certain embodiments, thecancer, tumor, growth abnormality, immune disorder, or microbialinfection may be characterized by cells that express (includingoverexpress) HER2/neu/ErbB2. The HER2/neu/ErbB2 may be expressed(including overexpressed) at the surface of the cells. Administration ofa “therapeutically effective dosage” of a composition of the presentinvention can result in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.The “patient in thereof” is as described herein. For certainembodiments, the “patient in need thereof” has been previously treatedwith one or more additional HER2-targeting therapeutic agent; and/or isundergoing treatment with one or more additional HER2-targetingtherapeutic agent. For certain embodiments, the “patient in needthereof” has been previously treated with one or more additionalHER2-targeting therapeutic agent is as described herein. For certainembodiments, the “patient in need thereof” is undergoing treatment withone or more additional HER2-targeting therapeutic agent is as describedherein. For certain embodiments, the “patient in need thereof” does notrespond to, or does not benefit from, treatment with one or moreadditional HER2-targeting therapeutic agent is as described herein. Theone or more additional HER2-targeting therapeutic agent is as describedherein. For example, the additional HER2-targeting therapeutic agent maycomprise a dual tyrosine kinase inhibitor; such as e.g. lapatinib and/orneratinib. For example, the additional HER2-targeting therapeutic agentmay comprise an anti-HER2 antibody that binds an antigenic determinantin HER2 that does not overlap with the antigenic determinant in HER2bound by the HER2-targeting molecule; such as e.g. T-DM1, trastuzumab,and/or pertuzumab. For example, the additional HER2-targetingtherapeutic agent may comprise anti-HER2 antibody drug conjugatetherapy; such as T-DM1. For certain embodiments, the one or moreadditional HER2-targeting therapeutic agent is selected from: lapatinib,neratinib, T-DM1, trastuzumab, and pertuzumab.

The therapeutically effective amount of a composition of the presentinvention will depend on the route of administration, the type oforganism being treated, and the physical characteristics of the specificpatient under consideration. These factors and their relationship todetermining this amount are well known to skilled practitioners in themedical arts. This amount and the method of administration can betailored to achieve optimal efficacy, and may depend on such factors asweight, diet, concurrent medication and other factors, well known tothose skilled in the medical arts. The dosage sizes and dosing regimenmost appropriate for human use may be guided by the results obtained bythe present invention, and may be confirmed in properly designedclinical trials. An effective dosage and treatment protocol may bedetermined by conventional means, starting with a low dose in laboratoryanimals and then increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g. topical administration of a cream, gel or ointment, or by means ofa transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including infraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

For administration of a pharmaceutical composition of the presentinvention, the dosage range will generally be from about 0.001 to 10milligrams per kilogram (mg/kg), and more, usually 0.001 to 0.5 mg/kg,of the subject’s body weight. Exemplary dosages may be 0.001 mg/kg bodyweight, 0.005 mg/kg body weight, 0.0075 mg/kg body weight, 0.015 mg/kgbody weight, 0.020 mg/kg body weight, or 0.025 mg/kg body weight orwithin the range of 0.001 to 0.030 mg/kg. Exemplary dosages may be 0.01mg/kg body weight, 0.03 mg/kg body weight, 0.05 mg/kg body weight, 0.075mg/kg body weight, or 0.1 mg/kg body weight or within the range of 0.01to 0.1 mg/kg. An exemplary treatment regime is a once or twice dailyadministration, or a once or twice weekly administration, once every twoweeks, once every three weeks, once every four weeks, once a month, onceevery two or three months or once every three to 6 months. Dosages maybe selected and readjusted by the skilled health care professional asrequired to maximize therapeutic benefit for a particular patient.

Pharmaceutical compositions of the present invention will typically beadministered to the same patient on multiple occasions. Intervalsbetween single dosages can be, for example, two to five days, weekly,monthly, every two or three months, every six months, or yearly.Intervals between administrations can also be irregular, based onregulating blood levels or other markers in the subject or patient.Dosage regimens for a composition of the present invention includeintravenous administration to a subject of 1 to 50 µg of HER2-targetingmolecule per kilogram (kg) body weight with the composition administeredonce or twice a week for three or more consecutive weeks, such as forfour or five weeks. Exemplary dosage regimens for a composition of thepresent invention include intravenous administration to a subject of 1to 25 µg of HER2-targeting molecule per kg body weight with thecomposition administered once or twice a week for three or moreconsecutive weeks, such as for four or five weeks. Dosage regimens for acomposition of the present invention include intravenous administrationto a subject of 10 to 50 µg of HER2-targeting molecule per kg bodyweight with the composition administered once or twice a week for threeor more consecutive weeks, such as for four or five weeks. Dosageregimens for a composition of the present invention include intravenousadministration of 0.01 to 1 mg/kg body weight or 0.03 to 3 mg/kg bodyweight with the composition administered every two to four weeks for sixdosages, then every three months at 0.01 to 3 mg/kg body weight or 0.01to 0.03 mg/kg body weight.

A pharmaceutical composition of the present invention may beadministered via one or more routes of administration, using one or moreof a variety of methods known in the art. As will be appreciated by theskilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration forcell-targeting molecules and pharmaceutical compositions of the presentinvention include, e.g. intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal, or other parenteral routes ofadministration, for example by injection or infusion. For otherembodiments, a cell-targeting molecule or pharmaceutical composition ofthe invention may be administered by a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually, or topically.

Therapeutic cell-targeting molecules or pharmaceutical compositions ofthe present invention may be administered with one or more of a varietyof medical devices known in the art. For example, in one embodiment, apharmaceutical composition of the invention may be administered with aneedleless hypodermic injection device. Examples of well-known implantsand modules useful in the present invention are in the art, includinge.g., implantable micro-infusion pumps for controlled rate delivery;devices for administering through the skin; infusion pumps for deliveryat a precise infusion rate; variable flow implantable infusion devicesfor continuous drug delivery; and osmotic drug delivery systems. Theseand other such implants, delivery systems, and modules are known tothose skilled in the art.

The cell-targeting molecule or pharmaceutical composition of the presentinvention may be administered alone or in combination with one or moreother therapeutic or diagnostic agents. A combination therapy mayinclude a cell-targeting molecule of the present invention, orpharmaceutical composition thereof, combined with at least one othertherapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, interalia, a cytotoxic, anti-cancer or chemotherapeutic agent, ananti-inflammatory or anti-proliferative agent, an antimicrobial orantiviral agent, growth factors, cytokines, an analgesic, atherapeutically active small molecule or polypeptide, a single chainantibody, a classical antibody or fragment thereof, or a nucleic acidmolecule which modulates one or more signaling pathways, and similarmodulating therapeutic molecules which may complement or otherwise bebeneficial in a therapeutic or prophylactic treatment regimen.

The cell-targeting molecule or pharmaceutical composition of the presentinvention may be administered alone or in combination with one or moreother HER2-targeting therapeutic agents. The cell-targeting molecule orpharmaceutical composition of the present invention may be administeredalone or in combination with one or more additional HER2-targetingtherapeutic agents, such as, e.g., T-DM1 (trastuzumab emtansine),trastuzumab, pertuzumab, and/or lapatinib. A combination therapy mayinclude a cell-targeting molecule of the present invention, orpharmaceutical composition thereof, combined with at least one othertherapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, interalia, a cytotoxic, anti-cancer or chemotherapeutic agent, ananti-inflammatory or anti-proliferative agent, an antimicrobial orantiviral agent, growth factors, cytokines, an analgesic, atherapeutically active small molecule or polypeptide, a single chainantibody, a classical antibody or fragment thereof, or a nucleic acidmolecule which modulates one or more signaling pathways, and similarmodulating therapeutic molecules which may complement or otherwise bebeneficial in a therapeutic or prophylactic treatment regimen. Forcertain embodiments, the methods of the invention for treating adisease, disorder, or condition in a patient in need thereof may furthercomprise administering to the patient a therapeutically effective amountof one or more additional HER2-targeting therapeutic agent. Theadditional HER2-targeting therapeutic agent is as described herein. Forexample, the additional HER2-targeting therapeutic agent may comprise adual tyrosine kinase inhibitor; such as lapatinib and/or neratinib. Forexample, the additional HER2-targeting therapeutic agent may comprise ananti-HER2 antibody that binds an antigenic determinant in HER2 that doesnot overlap with the antigenic determinant in HER2 bound by theHER2-targeting molecule; such as T-DM1, trastuzumab, and/or pertuzumabfor the HER2-targeting molecule which comprises, consists of, orconsists essentially of any one of SEQ ID NOs: 29, 36, 102, and 108. Forexample, the additional HER2-targeting therapeutic agent may compriseanti-HER2 antibody drug conjugate therapy; such as T-DM1.

Treatment of a patient with cell-targeting molecule or pharmaceuticalcomposition of the present invention preferably leads to cell death oftargeted cells and/or the inhibition of growth of targeted cells. Assuch, cytotoxic, cell-targeting molecules of the present invention, andpharmaceutical compositions comprising them, will be useful in methodsfor treating a variety of pathological disorders in which killing ordepleting target cells may be beneficial, such as, inter alia, cancer,tumors, other growth abnormalities, immune disorders, and infectedcells. The present invention provides methods for suppressing cellproliferation, and treating cell disorders involving HER2-expressingcells, including neoplasia.

In certain embodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention are for use in the treatment orprevention of a disease, disorder, or condition in a patient in needthereof. The disease, disorder or condition may be characterized bycells that are physically coupled with HER2 (e.g. the cells express HER2such that HER2 is expressed on the surfaces of the cells). In certainembodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention are for use in the treatment orprevention of a cancer, tumor (malignant and non-malignant), growthabnormality, immune disorder, and/or microbial infection in a patient inneed thereof. In certain embodiments, the cell-targeting molecules andpharmaceutical compositions of the present invention are for use in thetreatment or prevention of a cancer, tumor (malignant andnon-malignant), and/or growth abnormality in a patient in need thereof.In certain embodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention are for use in the treatment orprevention of a cancer and/or tumor (malignant and non-malignant) in apatient in need thereof. The cancer, tumor, growth abnormality, immunedisorder, and/or microbial infection may be characterized by cells thatare physically coupled with HER2 (e.g. the cells express HER2 such thatHER2 is expressed on the surfaces of the cells). The “patient inthereof” is as described herein. I n certain embodiments, the “patientin need thereof” has been previously treated with one or more additionalHER2-targeting therapeutic agent; and/or is undergoing treatment withone or more additional HER2-targeting therapeutic agent. For certainembodiments, the “patient in need thereof” has been previously treatedwith one or more additional HER2-targeting therapeutic agent is asdescribed herein. For certain embodiments, the “patient in need thereof”is undergoing treatment with one or more additional HER2-targetingtherapeutic agent is as described herein. For certain embodiments, the“patient in need thereof” does not respond to, or does not benefit from,treatment with one or more additional HER2-targeting therapeutic agentis as described herein. For certain embodiments, the treatment orprevention of a disease, disorder, or condition in a patient in needthereof may further comprise a step of administering to the patient atherapeutically effective amount of one or more additionalHER2-targeting therapeutic agent. The additional HER2-targetingtherapeutic agent is as described herein.

In certain embodiments, the present invention provides methods fortreating malignancies or neoplasms and other blood cell associatedcancers in a mammalian subject, such as a human, the method comprisingthe step of administering to a subject in need thereof a therapeuticallyeffective amount of a cytotoxic cell-targeting molecule orpharmaceutical composition of the present invention.

The cell-targeting molecules and pharmaceutical compositions of thepresent invention have varied applications. The cell-targeting moleculesand pharmaceutical compositions of the present invention are commonlyanti-neoplastic agents - meaning they are capable of treating and/orpreventing the development, maturation, or spread of neoplastic ormalignant cells by inhibiting the growth and/or causing the death ofcancer or tumor cells. However, certain embodiments of thecell-targeting molecule or pharmaceutical composition of the presentinvention is used to treat an immune disorder, such as, e.g., a T-cell-,B-cell-, plasma cell- or antibody- mediated disease or disorder.

Certain embodiments of the cell-targeting molecules and pharmaceuticalcompositions of the present invention can be utilized in a method oftreating cancer comprising administering to a patient, in need thereof,a therapeutically effective amount of a cell-targeting molecule and/orpharmaceutical composition of the present invention. For certainembodiments of the methods of the present invention, the cancer beingtreated is selected from the group consisting of: bone cancer (such asmultiple myeloma or Ewing’s sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi’s sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), anduterine cancer. For certain embodiments, the cancer to be treated isselected from the group consisting of: breast cancer, gastric cancer,urothelial cancer, bladder cancer, urothelial bladder cancer, serousuterine cancer, extrahepatic biliary tract cancer, and biliarycarcinoma. For certain embodiments, the cancer being treated is breastcancer and/or gastrointestinal cancer.

Among certain embodiments of the present invention is using the Shigatoxin effector polypeptide or cell-targeting molecule of the presentinvention as a component of a pharmaceutical composition or medicamentfor the treatment or prevention of a cancer, tumor, other growthabnormality, immune disorder, and/or microbial infection. For example,skin tumors may be treated with such a medicament in efforts to reducetumor size or eliminate the tumor completely.

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention to label or detect the interiors of neoplastic cells. Thismethod may be based on the ability of certain cell-targeting moleculesof the present invention to enter specific cell types and route withincells via retrograde intracellular transport, to the interiorcompartments of specific cell types are labeled for detection. This canbe performed on cells in situ within a patient or on cells and tissuesremoved from an organism, e.g. biopsy material.

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention to detect the presence of a cell type for the purpose ofinformation gathering regarding diseases, conditions and/or disorders.The disease, disorder, or condition may be characterized by cells thatare physically coupled with HER2. The HER2 target biomolecule may bephysically coupled to the surface of the cells. For certain embodiments,the disease, disorder or condition may be characterized by cells thatexpress the HER2 target biomolecule (including cells that overexpressHER2). The HER2 may be expressed (including overexpressed) at thesurface of the cells. The method comprises contacting a cell with adiagnostically sufficient amount of a cell-targeting molecule of thepresent invention in order to detect the molecule by an assay ordiagnostic technique. The phrase “diagnostically sufficient amount”refers to an amount that provides adequate detection and accuratemeasurement for information gathering purposes by the particular assayor diagnostic technique utilized. Generally, the diagnosticallysufficient amount for whole organism in vivo diagnostic use will be anon-cumulative dose of between 0.001 to 10 milligrams of the detectionpromoting agent linked cell-targeting molecule of the invention per kgof subject per subject. Typically, the amount of cell-targeting moleculeof the invention used in these information gathering methods will be aslow as possible provided that it is still a diagnostically sufficientamount. For example, for in vivo detection in an organism, the amount ofShiga toxin effector polypeptide, cell-targeting molecule, orpharmaceutical composition of the invention administered to a subjectwill be as low as feasibly possible.

The cell-type specific targeting of cell-targeting molecules of thepresent invention combined with detection promoting agents provides away to detect and image cells physically coupled with an extracellulartarget biomolecule of a binding region of the molecule of the invention.Imaging of cells using the cell-targeting molecules of the presentinvention may be performed in vitro or in vivo by any suitable techniqueknown in the art. Diagnostic information may be collected using variousmethods known in the art, including whole body imaging of an organism orusing ex vivo samples taken from an organism. The term “sample” usedherein refers to any number of things, but not limited to, fluids suchas blood, urine, serum, lymph, saliva, anal secretions, vaginalsecretions, and semen, and tissues obtained by biopsy procedures. Forexample, various detection promoting agents may be utilized fornon-invasive in vivo tumor imaging by techniques such as magneticresonance imaging (MRI), optical methods (such as direct, fluorescent,and bioluminescent imaging), positron emission tomography (PET),single-photon emission computed tomography (SPECT), ultrasound, x-raycomputed tomography, and combinations of the aforementioned (see, Kaur Set al., Cancer Lett 315: 97-111 (2012),for review).

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule, orpharmaceutical composition of the present invention in a diagnosticcomposition to label or detect the interiors of a hematologic cell,cancer cell, tumor cell, infected cell, and/or immune cell (see e.g.,Koyama Y et al., Clin Cancer Res 13: 2936-45 (2007); Ogawa M et al.,Cancer Res 69: 1268-72 (2009); Yang L et al., Small 5: 235-43 (2009)).Based on the ability of certain cell-targeting molecules of theinvention to enter specific cell types and route within cells viaretrograde intracellular transport, the interior compartments ofspecific cell types are labeled for detection. This can be performed oncells in situ within a patient or on cells and tissues removed from anorganism, e.g. biopsy material.

Diagnostic compositions of the present invention may be used tocharacterize a disease, disorder, or condition as potentially treatableby a related pharmaceutical composition of the present invention.Certain compositions of matter of the present invention may be used todetermine whether a patient belongs to a group that responds to atherapeutic strategy which makes use of a compound, composition orrelated method of the present invention as described herein or is wellsuited for using a delivery device of the invention.

Diagnostic compositions of the present invention may be used after adisease, e.g. a cancer, is detected in order to better characterize it,such as to monitor distant metastases, heterogeneity, and stage ofcancer progression. The phenotypic assessment of disease disorder orinfection can help prognostic and prediction during therapeutic decisionmaking. In disease reoccurrence, certain methods of the invention may beused to determine if local or systemic problem.

Diagnostic compositions of the present invention may be used to assessresponses to therapies regardless of the type of the type of therapy,e.g. small molecule drug, biological drug, or cell-based therapy. Forexample, certain embodiments of the diagnostics of the invention may beused to measure changes in tumor size, changes in antigen positive cellpopulations including number and distribution, or monitoring a differentmarker than the antigen targeted by a therapy already being administeredto a patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6(2004); Evans M et al., Proc. Natl. Acad. Sci. USA 108: 9578-82 (2011)).

For certain embodiments of the method used to detect the presence of acell type may be used to gather information regarding diseases,disorders, and conditions, such as, for example bone cancer (such asmultiple myeloma or Ewing’s sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi’s sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), uterinecancer, acute lymphoblastic leukemia (ALL), T acute lymphocyticleukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloidleukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B-cellprolymphocytic lymphoma, Burkitt’s lymphoma (BL), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloidleukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma,hairy cell leukemia (HCL), Hodgkin’s Lymphoma (HL), intravascular largeB-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacyticlymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM),natural killer cell leukemia, nodal marginal B-cell lymphoma,Non-Hodgkin’s lymphoma (NHL), plasma cell leukemia, plasmacytoma,primary effusion lymphoma, pro-lymphocytic leukemia, promyelocyticleukemia, small lymphocytic lymphoma, splenic marginal zone lymphoma,T-cell lymphoma (TCL), heavy chain disease, monoclonal gammopathy,monoclonal immunoglobulin deposition disease, myelodysplastic syndromes(MDS), smoldering multiple myeloma, and Waldenstrom macroglobulinemia.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, or pharmaceuticalcompositions thereof, are used for both diagnosis and treatment, or fordiagnosis alone. In some situations, it would be desirable to determineor verify the HLA variant(s) and/or HLA alleles expressed in the subjectand/or diseased tissue from the subject, such as, e.g., a patient inneed of treatment, before selecting a cell-targeting molecule of theinvention for use in treatment(s).

Any embodiment of the Shiga toxin effector polypeptide of the presentinvention and cell-targeting molecule of the present invention (e.g.embodiments of embodiment Sets #1-3 in the Summary) may be used witheach individual embodiment of the methods of the present invention.

The present invention is further illustrated by the followingnon-limiting examples of 1) Shiga toxin effector polypeptides of thepresent invention, 2) cell-targeting molecules of the present invention,and 3) cytotoxic, cell-targeting molecules of the present inventioncomprising the aforementioned polypeptides and capable of specificallytargeting certain cell types.

EXAMPLES

The following examples demonstrate certain embodiments of the presentinvention. However, it is to be understood that these examples are forillustration purposes only and do not intend, nor should any beconstrued, to be wholly definitive as to conditions and scope of thisinvention. The experiments in the following examples were carried outusing standard techniques, which are well known and routine to those ofskill in the art, except where otherwise described.

The following examples describe several, exemplary, cytotoxic, Shigatoxin A Subunit derived polypeptide scaffolds comprising Shiga toxineffector polypeptides of the present invention. The Shiga toxin effectorpolypeptides in the Examples are de-immunized while retaining potentcytotoxic activities.

The following examples also describe several, cytotoxic, cell-targetingmolecules, each molecule comprising a Shiga toxin effector polypeptidelinked, either directly or indirectly, to a cell-targeting bindingregion capable of specifically binding an extracellular part of a HER2target biomolecule physically associated with a cellular surface of acell. Exemplary, cytotoxic, cell-targeting molecules described belowbound to cell-surface, target biomolecules expressed by targeted, tumorcell-types and entered those targeted cells. The internalized,cell-targeting molecules effectively routed their Shiga toxin effectorpolypeptides to the cytosols of target cells where the Shiga toxineffector polypeptides inactivated ribosomes and subsequently caused theapoptotic death of the targeted cells.

Additionally, some of the exemplary cell-targeting molecules compriseprotease-cleavage resistant, de-immunized, Shiga toxin effectorpolypeptides that exhibit improved in vivo immunogenicity profiles(reductions in antibody responses) as compared to parental cytotoxicmolecules comprising a furin-cleavage resistant, Shiga toxin effectorpolypeptide that has not been further de-immunized by the disruption ofadditional, endogenous epitope regions. Furthermore, these exemplary,protease-cleavage resistant, de-immunized cell-targeting moleculesexhibit improved in vivo tolerability as compared to relatedcell-targeting molecules comprising more protease-cleavage sensitiveShiga toxin effector polypeptide regions.

The Examples below describe certain, cell-targeting molecules of thepresent invention and their properties. Certain Examples describecell-targeting molecules of the present invention wherein a Shiga toxineffector polypeptide component (1) is de-immunized; (2) is on orproximal to an amino-terminus of a polypeptide component of thecell-targeting molecule; (3) is furin-cleavage resistant; and/or (4)comprises an embedded or inserted T-cell epitope.

Example 1. HER2-Targeting Molecules Comprising Furin-Cleavage Resistant,Shiga Toxin A Subunit Derived Polypeptides

Various HER2-targeting molecules, each comprising (1) at least oneimmunoglobulin-type binding region targeting HER2 and (2) at least oneShiga toxin A Subunit effector polypeptide were constructed and testedfor use in killing HER2-positive cancer cells.

A. Construction and Production of HER2-Targeting Molecules

Cytotoxic, cell-targeting molecules were designed to target HER2 usingvarious Shiga toxin A Subunit effector polypeptides (each capable ofproviding one or more Shiga toxin A Subunit functions) and variousimmunoglobulin-type binding regions, each capable of binding anextracellular part of human HER2, as cell-targeting binding regions. Theimmunoglobulin-type binding region of these HER2-targeting molecules waseither a single-chain antibody variable fragment or a camelid V_(H)Hthat binds with high-affinity, specificity, and selectivity to acell-surface HER2 target biomolecule physically coupled to the surfaceof human cancer cells. Polynucleotides were constructed which encodefusion proteins comprising the aforementioned components: (1) at leastone anti-HER2 antibody variable fragment and (2) at least one Shigatoxin A Subunit effector polypeptide. These polynucleotides were used toproduce cytotoxic, cell-targeting molecules of the present invention,including 114773 (SEQ ID NO:22), 115172 (SEQ ID NO:23), 114778 (SEQ IDNO:24), 114795 (SEQ ID NO:25), 114791 (SEQ ID NO:26), 114912 (SEQ IDNO:28), 115111 (SEQ ID NO:29), 115411 (SEQ ID NO:30), 114898 (SEQ IDN031), 115195 (SEQ ID NO:32), 115194 (SEQ ID NO:33), 115645 (SEQ IDNO:34), and 115845 (SEQ ID NO:35). All of the cell-targeting moleculestested in the experiments of this Example, including referencecell-targeting molecules, were produced in a bacterial system andpurified by column chromatography using techniques well-known to theskilled worker. The purification of certain exemplary HER2-targetingmolecules of the present invention was facilitated by the use of a fusedaffinity tag, such as, e.g., a chitin-binding domain (SEQ ID NO:43) or a6xHis polyhistidine tag (SEQ ID NO:44).

1. Chitin Affinity Based Purification

For certain exemplary HER2-binding proteins of this Example, cloning andpurification were done essentially as described in the manufacturer’smanual for the IMPACT™ (Intein Mediated Purification with an AffinityChitin-binding Tag) system (New England Biolabs, Ipswich, MA, U.S.A.).An affinity tag used to purify some of the HER2-targeting molecules ofthis Example was the intein chitin binding domain (CBD) sequence (SEQ IDNO:43), which was fused to the carboxy-terminals of some of the fusionproteins of this Example using the E. coli expression vector pTxb1 (NewEngland Biolabs, Ipswich, MA, U.S.A.). These CBD fusion proteins wereexpressed in bacteria, extracted from the soluble fraction, and thenallowed to bind to a chitin column. The intein was then cleaved awayfrom the fusion protein by incubation with dithiolthreitol (DTT), andthe HER2-binding proteins of interest were eluted away from the chitincolumn after removal of the CBD affinity tag (SEQ ID NO:43).

Exemplary HER2-targeting fusion proteins of the present invention 114778(SEQ ID NO:24), 114795 (SEQ ID NO:25), and 114791 (SEQ ID NO:26) wereexpressed and samples were analyzed by SDS-PAGE (FIG. 2 ). All three ofthese protein samples were predominantly comprised by a protein speciesof about 55 kDa as measured by SDS-PAGE in reducing conditions (FIG. 2).

These exemplary HER2-targeting molecules were then tested for cytotoxicactivity using the following cytotoxicity assay. Certain human tumorcell-line cells were plated in 20 µL cell culture medium in 384-wellplates (typically at 1-2 x 10³ cells per well for adherent cells, platedthe day prior or day of addition of HER2-targeting molecule). A seriesof dilutions (typically 10-fold) of the molecules to be tested wasprepared in an appropriate buffer, and 5 µL of the dilutions orbuffer-only control were added to the plated cells. Control wellscontaining only cell culture medium were used for baseline correction.The cell samples were incubated with the HER2-targeting molecule or justbuffer for 3 or 5 days at 37° C. and in an atmosphere of 5% carbondioxide (CO₂). The total cell survival or percent viability wasdetermined using a luminescent readout using the CellTiter-Glo®Luminescent Cell Viability Assay (Promega Corp., Madison, WI, U.S.A.)according to the manufacturer’s instructions. The human cells testedincluded cells from the HCC1954 and NCI/ADR-RES cell lines, wherecertain samples of NCI/ADR-RES cells were transfected with a HER2expression vector to make them express HER2 to a cell-surface insufficient quantities to make them HER2 positive (referred to herein as“NCI/ADR-RES-HER2+”).

The Percent Viability of cells in experimental wells was calculatedusing the following equation: (Test RLU - Average Media RLU) ÷ (AverageCells RLU - Average Media RLU) × 100. The logarithm of thecell-targeting molecule protein concentration versus Percent Viabilitywas plotted in Prism (GraphPad Prism, San Diego, CA, U.S.A.) and log(inhibitor) versus response (3 parameter) analysis or and log(inhibitor) versus normalized response analysis were used to determinethe half-maximal cytotoxic concentration (CD₅₀) value for the testedmolecule. The CD₅₀ value(s) for each molecule tested were calculated,when possible. When CD₅₀ values could not be calculated based on theshape of the curve over the concentrations tested, then a CD₅₀ value wasnoted as being beyond the maximum tested concentration. All graphs andnon-linear regressions were done with GraphPad Prism and flow cytometrydata was analyzed with FloJo software.

Results of the cytotoxicity assay are reported below (see Table 1 andFIG. 3 ). The exemplary HER2-targeting molecules 114778 (SEQ ID NO:24),114795 (SEQ ID NO:25), and 114791 (SEQ ID NO:26) were cytotoxic to HER2positive cells (Table 1; FIG. 3 ). In some experiments, HER2 negativecells were also treated with the maximum concentration of theHER2-targeting molecule in the dilution series, and, under theseconditions, the HER2 negative cells did not show any change in viabilityas compared to a buffer only control.

TABLE 1 Cytotoxicities of Exemplary HER2-Targeting Molecules of thePresent Invention Purified Using a Chitin-Binding Affinity Tag andIntein-Mediated Cleavage Away from the Tag Purification Method: Chitinbinding via intein tag and tag cleavage with DTT CD₅₀ (ng/mL) CD₅₀(ng/mL) HER2-Targeting Molecule HER2 positive HCC1954 cells HER2positive NCI/ADR-RES cells 114778 36.9 26.4 114791 20.9 17.1 114795 14.95.5

This data demonstrated similar cytotoxic potencies among 114795 (SEQ IDNO:25), 114778 (SEQ ID NO:24), and 114791 (SEQ ID NO:26), all of whichwere fusion proteins purified using the IMPACT™ CBD intein affinity tag,chitin-binding purification system.

2. Protein L Affinity Based Purification

An alternative method of protein purification based on Protein L bindingaffinity was used and compared to the intein-CBD affinity tag methodused above involving the IMPACT™ system. The binding affinity betweenbacterial Protein L and certain scFv’s was used to purify exemplaryHER2-targeting molecules of the present invention: 114773 (SEQ ID NO:22)comprising a carboxy-terminal intein-CBD tag (SEQ ID NO:43), 114912 (SEQID NO:28), 115111 (SEQ ID NO:29), and 115411 (SEQ ID NO:30). FIGS. 4-5show SDS-PAGE analyses of samples of 114773 (SEQ ID NO:22), 114791 (SEQID NO:26), 114912 (SEQ ID NO:28), 115111 (SEQ ID NO:29), and 115411 (SEQID NO:30) after purification using a Protein L binding affinity method.

FIG. 4 shows an SDS-PAGE analysis of 114773 (SEQ ID NO:22) (with acarboxy-terminal intein-CBD tag (SEQ ID NO:43)) and 114791 (SEQ IDNO:26) (with a carboxy-terminal intein-CBD tag (SEQ ID NO:43)) samplesafter purification using a Protein L binding affinity method. FIG. 5shows SDS-PAGE analysis of 114912 (SEQ ID NO:28) (without any intein-CBDtag), 115111 (SEQ ID NO:29) (without any intein-CBD tag), and 115411(SEQ ID NO:30) (without any intein-CBD tag).

Exemplary HER2-targeting molecules 114912 (SEQ ID NO:28) and 115111 (SEQID NO:29) purified using Protein L binding were tested for cytotoxicactivity using the assay as described above for the samples purifiedusing the CBD intein system. The results of the cytotoxicity assay arereported below (see Table 2 and FIG. 6 ).

TABLE 2 Cytotoxicities of Exemplary HER2-Targeting Molecules of thePresent Invention Purified Using Protein L Binding Affinity PurificationMethod: Protein L binding via scFv CD₅₀ (ng/mL) HER2-targeting moleculeHER2 positive HCC1954 cells HER2 positive NCI/ADR-RES cells HER2positive JIMT-1 cells HER2 positive SK-OV-3 cells HER2 positive HCC1419cells HER2 negative JIMT-1 cells 114912 9.0 14.0 78.9 43.2 33.3 >2,000115111 1.6 2.8 5.9 6.4 11.8 >2,000

The exemplary HER2-targeting fusion proteins 114912 (SEQ ID NO:28) and115111 (SEQ ID NO:29) were cytotoxic to HER2 positive cells (Table 2;FIG. 6 ). This data demonstrated the greater cytotoxic potency of 115111(SEQ ID NO:29) as compared to 114912 (SEQ ID NO:28), both of which werepurified using Protein L affinity. No cytotoxicity toward MCF-7 cells,which express very low levels of HER2, was observed for most of theHER2-targeting molecule concentrations tested (FIG. 6 ).

Additional exemplary HER2-targeting molecules of the present inventionthat are related to 115111 (SEQ ID NO:29) were tested for cytotoxicactivities toward HER2 positive cell lines using the cytotoxicity assaydescribed above. The proteins 115172 (SEQ ID NO:23), 115195 (SEQ IDNO:32), and 115194 (SEQ ID NO:33) are related to 115111 (SEQ ID NO:29)because they each comprise identical heavy and light variable domains.

The skilled worker will appreciate that the length of the linker betweenvariable domains (or “interdomain linker”) in a scFv can affect thespontaneous assembly of non-covalent, multimeric, multivalent molecules.Generally, linkers that are between three amino acid residues and twelveamino acid residues in length (e.g. the pentamer G₄S (SEQ ID NO:94))promote diabody formation via intermolecular variable domain swapping;whereas longer linkers (e.g. (G₄S)₅ (SEQ ID NO:92)) allow forintramolecular heavy and light chain pairing, resulting in predominantlymonomeric molecules (see e.g. WO 2018/140427). 115111 (SEQ ID NO:29)comprises a 25-mer interdomain linker and was verified to predominantlyform monovalent monomers. 115195 (SEQ ID NO:32) comprises a pentamerinterdomain linker and was verified to predominantly form divalentdimers. 115194 (SEQ ID NO:33) comprises an identical scFv to 115195 (SEQID NO:32) having the same pentamer interdomain linker and is predictedto form divalent dimers like 115195 (SEQ ID NO:32). 115172 (SEQ IDNO:23) and 115194 (SEQ ID NO:33) differ from 115111 (SEQ ID NO:29) and115195 (SEQ ID NO:32) in that its Shiga toxin A Subunit effectorpolypeptide SLTA-FR (SEQ ID NO:37) comprises mostly wild-type sequences,having mutations only in the minimal furin-cleavage site at thecarboxy-terminus of the A1 fragment and disrupting Epitope Region #8(Table B, supra). Cytotoxicity data for these molecules are reportedbelow (see Table 3 and FIG. 7 .

TABLE 3 Cytotoxicity of 115111 and Related HER2-Targeting Molecules CD₅₀(ng/mL) Cell Line Cancer Type HER2 Expression 115111 115195 115172115194 HCC1954 breast high 4.6 5.6 1.5 3.1 NCI/ADR-RES-HER2+ ovarian,transfected with HER2 high 5.1 3.6 1.8 1.8 HCC1569 breast high 21.1(45%) 17.2 (55%) 9.1 (25%) 9.9 (35%) JIMT-1 breast medium 5.9 (35%) 6.7(37%) 3.3 (21%) 6.8 (22%) ST486 lymphoma negative ~ 5,000 ~ 5,000 ~5,000 ~ 5,000

The data from this cytotoxicity experiment indicated that the monomer115111 (SEQ ID NO:29), the dimer 115195 (SEQ ID NO:32), the predictedmonomer 115172 (SEQ ID NO:23), and the predicted dimer 115194 (SEQ IDNO:33) all exhibited similar cytotoxic activities in vitro (see e.g.Table 3 and FIG. 7 ). No cytotoxicity toward HER2 negative cells wasobserved for most of the HER2-targeting molecule concentrations tested(e.g. at concentrations below 100 ng/mL).

B. Testing In Vitro Activities of Exemplary HER2-Targeting Molecules ofthe Present Invention 1. Ribosome Inhibition Activities

Exemplary HER2-targeting molecules of the present invention 115111 (SEQID NO:29) and 115411 (SEQ ID NO:30) were tested for enzymatic activityafter purification using Protein L binding as described above. Theircatalytic activities regarding ribosome inactivation were compared withthe de-immunized SLT-1A1 fragment alone (DI-2 (SEQ ID NO:20)) and theHER2-targeting molecule 115172 (SEQ ID NO:23) comprising a mostlywild-type SLT-1A sequence having alterations only to mutate the furincleave motif (SLTA-FR) and disrupt Epitope Region #8 (Table B, supra)(SEQ ID NO:37).

The ribosome inhibition assay used a cell-free, in vitro proteintranslation assay using the TNT® Quick Coupled Transcription/Translationkit (L1170 Promega Madison, WI, U.S.A.). The kit includes Luciferase T7Control DNA (L4821 Promega Madison, WI, U.S.A.) and TNT® Quick MasterMix. The ribosome activity reaction was prepared according tomanufacturer instructions. A series (typically 10-fold) of dilutionswere prepared in appropriate buffer and a series of identical TNTreaction mixture components were created for each dilution. The proteinsamples were combined with each of the TNT reaction mixtures along withthe Luciferase T7 Control DNA. The test samples were incubated for 1.5hours at 30° C. After the incubation, Luciferase Assay Reagent (E1483Promega, Madison, WI, U.S.A.) was added to all test samples and theamount of luciferase protein translation was measured by luminescenceaccording to the manufacturer instructions. The level of translationalinhibition was determined by non-linear regression analysis oflog-transformed concentrations of total protein versus relativeluminescence units. Using statistical software (GraphPad Prism, SanDiego, CA, U.S.A.), the half maximal inhibitory concentration (IC₅₀)value was calculated for each sample using the Prism software functionof log(inhibitor) vs. response (three parameters) [Y = Bottom + ((Top -Bottom) / (1+10^(X - Log IC₅₀)))] under the headingdose-response-inhibition. The results of the ribosome inhibition assayare reported below (see Table 4 and FIG. 8 ).

TABLE 4 Ribosome Inhibition Activities and Cytotoxicities of ExemplaryHER2-Targeting Molecules Purified Using Protein L Binding AffinityRibosome Inhibition Cytotoxicity HER2-Targeting Molecule IC₅₀ (pM) CD₅₀HER2 positive NCI/ADR-RES cells 115172 32.01 0.77 ng/mL 115111 24.201.15 ng/mL 115411 6.60 4.59 ng/mL SLTA-DI-2 20.27 > 2,000 ng/mL

The exemplary HER2-targeting fusion proteins of the present invention115172 (SEQ ID NO:23), 115111 (SEQ ID NO:29), and 115411 (SEQ ID NO:30)inhibited ribosomes with high potency, e.g. in the low picomolar (pM)range (see Table 4; FIG. 8 ). The IC₅₀ value for ribosome inhibitionmeasured for 115111 (SEQ ID NO:29) was 24.2 pM (Table 4). The similarlevels of protein ribosome inhibition exhibited by the Shiga toxineffector polypeptide SLTA-DI-2 (SEQ ID NO:20) and the exemplaryHER2-targeting molecules 115172 (SEQ ID NO:23), 115111 (SEQ ID NO:29)and 115411 (SEQ ID NO:30) demonstrated these molecules retained theexpected mechanism of action (catalytic inhibition of protein synthesis)as fusion proteins wherein the Shiga toxin A Subunit effectorpolypeptide component of either 115111 (SEQ ID NO:29) or 115411 (SEQ IDNO:30) or 115172 (SEQ ID NO:23),was fused to an immunoglobulin-typeHER2-targeting binding region.

The exemplary HER2-targeting molecules 115172 (SEQ ID NO:23), 115111(SEQ ID NO:29), and 115411 (SEQ ID NO:30) were also tested for cytotoxicactivity toward HER2 positive NCI/ADR-RES cells transfected with HER2 asdescribed above and the results are reported here, in Table 4 above, andin FIG. 9 . The cytotoxic activities of these exemplary HER2-targetingfusion proteins were compared with the de-immunized SLT-1A1 fragmentalone (SLTA-DI-2 (SEQ ID NO:20)) and the HER2-targeting molecule 115172(SEQ ID NO:23) comprising a Shiga-like toxin A1 fragment SLTA-FR (SEQ IDNO:37) having alterations to mutate the furin cleavage motif and disruptEpitope Region #8 (Table B, supra). The cytotoxicity data from thisexperiment demonstrated that the HER2-targeting moiety was necessary tokill target cells, as there was no cytotoxic effect at concentrations upto 2,000 µg/mL with the untargeted SLTA-DI-2 construct (SEQ ID NO:20)(see FIG. 9 ). The cytotoxicity CD₅₀ value measured for 115111 (SEQ IDNO:29) was 1.2 ng/mL to NCI/ADR-RES-HER2+ cells.

2. HER2 Binding Activities

To investigate the cell-binding properties of exemplary HER2-targetingmolecules of the present invention, the binding activities ofHER2-targeting molecules to human HER2 positive HCC1954 cells weremeasured using a flow cytometry-based method. A dilution series of eachHER2-targeting molecule being tested was added to cells, the cells werewashed, and cell-bound HER2-targeting protein was detected using ananti-SLTA-DI-2 monoclonal antibody (mAb) conjugated to FITC, whichrecognizes de-immunized Shiga toxin effector polypeptides (e.g. SEQ IDNO:20). The FITC signal was measured using flow cytometry to generatemean fluorescent intensity (MFI) values for each sample. The MFI valueswere plotted as a function of log transformed HER2-targeting moleculeconcentration to determine the Bmax and K_(D) using a non-linear curveregression analysis (Prism (GraphPad Prism, San Diego, CA, U.S.A., Prismsoftware function sigmoidal 4PL). Results of this study are reportedbelow (see Table 5 and FIG. 10 ).

TABLE 5 Binding of HER2-Targeting Molecules to HER2 Positive HCC1954Cells HER2-Targeting Molecule K_(D) (µg/mL) Bmax (MFI) 114912 1.28 2119115111 0.42 2747 115845 0.32 2707 115195 0.30 2447 115645 0.25 1327

The exemplary HER2-targeting fusion proteins of the present invention115111 (SEQ ID NO:29), 115845 (SEQ ID NO:35), and 115195 (SEQ ID NO:32)exhibited similar binding in vitro to HCC1954 cells. 115111 (SEQ IDNO:29) bound HER2 positive HCC1954 cells with a K_(D) of 0.42 µg/mL, and115845 bound HER2 positive HCC1954 cells with a K_(D) of 0.32 µg/mL. Inthis binding assay, 114912 (SEQ ID NO:28) exhibited slightly lessbinding affinity to HCC1954 cells compared to the other HER2-targetingmolecules tested. In this assay, 115645 (SEQ ID NO:34) exhibited asimilar K_(D) but had a reduced Bmax compared to the otherHER2-targeting molecules tested.

A. HER2 Epitope Mapping of Binding by Exemplary HER2-Targeting Molecule115111

The extracellular epitope of human HER2 bound by 115111 (SEQ ID NO:29)was mapped to identify contact residues using a shotgun mutagenesismethod by Integral Molecular (Philadelphia, PA, U.S.A.). To determinethe optimal concentration of 115111 (SEQ ID NO:29) for screening HER2epitope binding, HEK-293T cells were transfected with a vector designedto express a wild-type (WT) construct of a target human HER2 protein(UniProt ID: P04626 (SEQ ID NO:38)) or with vector alone in a 384-wellformat. Serial dilutions of 115111 (SEQ ID NO:29) were tested viahigh-throughput flow cytometry for immunoreactivity against cellsexpressing the WT HER2 target protein (SEQ ID NO:38) or vector alone.The optimal screening concentration for 115111 (SEQ ID NO:29) for thisbinding assay was determined based on the raw signal values andsignal-to-background calculations. The same process was conducted todetermine the optimal screening concentration for trastuzumab, a controlHER2-binding antibody.

An alanine scanning mutagenesis library based on the human HER2 protein(SEQ ID NO:38) mentioned above was created and screened for 115111 (SEQID NO:29) binding in duplicate by high-throughput flow cytometry. Foreach test point, background fluorescence was subtracted from the rawdata, which were then normalized to 115111 (SEQ ID NO:29) binding withWT HER2 protein (SEQ ID NO:38). Critical mutagenesis clones wereidentified related to the binding of 115111 (SEQ ID NO:29) to WT HER2(SEQ ID NO:38). The primary critical residues for 115111 (SEQ ID NO:29)binding to HER2 (SEQ ID NO:38) were residues whose mutations werereduced for 115111 (SEQ ID NO:29) binding when tested with 115111 (SEQID NO:29) but were similar to WT HER2 target protein control when testedfor trastuzumab binding (a HER2-binding, positive control mAb). Theresulting mean binding reactivity values for selected critical residuepositions in WT human HER2 (SEQ ID NO:38) expressed as a percentage ofWT HER2 binding with 115111 (SEQ ID NO:29) measured using this epitopemapping assay are listed in Table 6.

TABLE 6 Certain Critical and Secondary Residues in HER2 Involved inBinding 115111 Binding Reactivity (% WT) HER2 mutation 115111trastuzumab Y112A 9.2 87.7 Q178A 34.9 119.5 L181A 20.1 95.8 G152A 39.289.7

Using the approach above, the HER2 epitope bound by 115111 (SEQ IDNO:29) appeared to comprise Y112, Q178, and L181, which all map todomain I of the HER2 extracellular domain (ECD) (NCBI accessionsNP_004439.2, amino acid residues 23 to 653) (SEQ ID NO:39). One residue,G152, was identified in the epitope mapping assay as a secondary residuein that it did not meet the threshold guidelines for being ‘critical’,but the G152A mutation did result in a large decrease of 115111 (SEQ IDNO:29) binding compared to the WT HER2 (SEQ ID NO:38). Binding to theG152A expressing clone showed a level of binding that was 39% of WTbinding as opposed to the 35% threshold level required to categorizeG152 a primary critical residue. The G152 position in HER2 is in closeproximity to the primary critical residues mapped using the assay, whichsuggested that it may also be part of the epitope in human HER2 (SEQ IDNO:38) bound by 115111 (SEQ ID NO:29).

FIG. 11 shows a diagram of human HER2 with markings to highlight thecritical and secondary residues for binding by 115111 (SEQ ID NO:29) asmeasured by the approach described above. In FIG. 11 on the left,primary residues critical for 115111 (SEQ ID NO:29) binding to HER2 (SEQID NO:38) are visualized as red spheres based on a crystal structure ofthe HER2 (PDB 1S78, Franklin M et al., Cancer Cell 5: 317-28 (2004))along with a secondary residue visualized as a blue sphere. In FIG. 11on the right, the critical HER2 binding residues for 115111 (SEQ IDNO:29) binding are shown as blue spheres, for pertuzumab binding areshown as magenta spheres, and for trastuzumab binding are shown aspurple spheres based on the data above and information in Cho H et al.,Nature 421: 756-60 (2003); Franklin M et al., Cancer Cell 5: 317-28(2004). The HER2 epitope bound by 115111 (SEQ ID NO:29) was mappedwithin the HER2 extracellular domain (ECD) (SEQ ID NO:39) to domain I;in contrast, pertuzumab binds to an epitope mapped to domain II of theECD of HER2 and trastuzumab binds to an epitope mapped to domain IV ofthe ECD of HER2 (SEQ ID NO:39) (see FIG. 11 ). Thus, 115111 (SEQ IDNO:29) binds an epitope in HER2 that is different from the epitopesbound by trastuzumab and pertuzumab.

B. HER2 Binding Specificity of Exemplary HER2-Targeting Molecule 115111

The binding specificity, affinity, and selectivity of the exemplaryHER2-targeting fusion protein of the present invention 115111 (SEQ IDNO:29) were tested by analyzing binding of this purified fusion proteinto a membrane proteome array, which comprised 5,300 different proteinstransfected to be expressed on the cell surface of HEK-293T cells(Integral Molecular, Inc., Philadelphia, PA, U.S.A.). The results shownin FIG. 12 show that only HER2 was identified and validated among the5,300 proteins as a selective binding target of 115111 (SEQ ID NO:29).

3. Cytotoxic Activities 1. Cytotoxic Activities In Vitro Compared toOther Drugs

The cytotoxicity of 115111 (SEQ ID NO:29) to HER2 positive cells wasevaluated in comparison to and in combination with referenceHER2-targeted therapeutics, including T-DM1 (trastuzumab emtansine),trastuzumab, and pertuzumab. As all of these therapeutic moleculesspecifically bind to an extracellular part of human HER2, thesemolecules might compete for HER2 binding with each other or otherwiseinteract so as to alter their functional activities when combined. Forexample, when different, cytotoxic, HER2-targeted molecules areadministered in combination, either a reduction or increase in cytotoxicactivities toward HER2 positive cells might be observed. In a furtherexample, when different, cytotoxic, HER2-targeted molecules areadministered in combination, an increase in the death of HER2-expressingcells might be observed as result of additive or synergistic effects oncytotoxicity.

To investigate the number of possible HER2 receptors on the cellsurfaces of cells of different cell types, an experiment was performedto quantify the number of HER2-targeted antibody molecules bound percell. This experiment involved the incubation of cells with an anti-HER2antibody conjugated to phycoerythrin (PE) (anti-HER2-PE, clone 24D2,Biolegend, San Diego, CA, U.S.A.). Then the samples were analyzed usingflow cytometry to quantify binding. Standard curves generated with BDQuantibrite™ PE beads having known PE loads were used to convert MFIsignal values to the number of antibodies bound per cell for eachsample. These HER2 positive cells were then used in cytotoxicity assaysperformed essentially as described above to compare the cytotoxicactivities of 115111 (SEQ ID NO:29) to T-DM1 and lapatinib. Results fromthese cytotoxicity assays are reported below and in Table 7, and arepresentative data set for T-DM1 is shown in FIG. 13 .

TABLE 7 HER2 Positive Cell Binding Sites per Cell and In Vitro CytotoxicActivities of 115111 as Compared to T-DM1 and Lapatinib HER2 PositiveCell Binding In Vitro Cytotoxicity CD₅₀ Cell Line cancer type antibodiesbound per cell 115111 T-DM1 lapatinib HCC1954 breast 2,250,000 1.39ng/mL 8.98 ng/mL 786 nM NCI-N87 gastric 2,270,000 3.76 ng/mL 18.48 ng/mL112 nM NCI/ADR-RES-HER2+ ovarian, transfected with HER2 1,340, 000 1.17ng/mL no viability change 2,704 nM HCC1569 breast DNT 2.17 ng/mL 13.66ng/mL 885 nM HCC1419 breast 3,070,000 6.58^(∗) ng/mL 74.56^(∗) ng/mL DNTAU565 breast DNT 7.04 ng/mL 0.63 ng/mL DNT JIMT-1 breast 307,0006.55^(∗) ng/mL 248.7^(∗) ng/mL 5,650 nM ‘^(∗)’ Indicates that cellviability plateaus above 20% in this assay; DNT = did not test

115111 (SEQ ID NO:29) exhibited potent cytotoxicity to all HER2+ cellstested in this experiment: HCC1954, NCI-N87, HCC1569, HCC1419, AU565,and JIMT-1 cells (Table 7; see also Tables 2-3, 8-10; FIGS. 6-7, and13-19 ).

Both 115111 (SEQ ID NO:29) and T-DM1 were potently cytotoxic toHCCC1954, NCI-N87, HCC1569, HCC1419, and AU565 cells (Table 7), with115111 exhibiting higher cytotoxic potency than T-DM1 to HCCC1954,NCI-N87, HCC1569, and HCC1419 cells (Table 7; see e.g. FIG. 13 ). 115111(SEQ ID NO:29) exhibited more potent cytotoxicity than T-DM1 towardNCI/ADR-RES-HER2+ and JIMT-1 cells (Table 7; see FIG. 13 ).

NCI/ADR-RES cells are considered resistant to T-DM1 due to MDR1expression. Because 115111 (SEQ ID NO:29) killed HER2 positiveNCI/ADR-RES cells (Tables 2-3 and 7; FIGS. 6-7, 9, and 13 ), 115111 (SEQID NO:29) is likely to be cytotoxic to other HER2 positive cellsexpressing p-glycoprotein 1 (Pgp) type multidrug resistance effluxpumps.

JIMT-1 cells are considered resistant to trastuzumab due to epitopemasking via MUC-4 and/or CD44 expression (see e.g. Wilken J, Maihle N,Ann N Y Acad Sci 1210: 53-65 (2010)). Because 115111 (SEQ ID NO:29)killed JIMT-1 cells (Tables 1-2 and 7; FIGS. 6-7 ), the presence ofMUC-4 and/or CD44 does not prevent 115111 (SEQ ID NO:29) cytotoxicity,such as, e.g., via epitope masking (see e.g. FIGS. 6-7 and Table 7).

As 115111 (SEQ ID NO:29) binds to an extracellular part of HER2 andinternalizes into target cells to seeking out their ribosomes forinactivation, the cytotoxicity activity of 115111 (SEQ ID NO:29) is notdependent on the binding to or inactivation of a kinase domain of HER2.In contrast, lapatinib is a dual tyrosine kinase inhibitor which targetsHER2 and EGFR by binding to their kinase domains and inhibiting kinaseactivity. Cancer therapies relying on drugs like lapatinib may berendered less effective due to the development of resistance mechanisms,such as protective mutations in the kinase catalytic domain of HER2 orde novo aberrations resulting in constitutive/unregulatedHER2-signaling-pathway activation or overactivation of the HER2signaling pathway at a point downstream of HER2 thereby bypassing thereceptor, such as in the absence of HER2 kinase activity or even HER2expression. Thus, certain HER2-targeting molecules of the presentinvention (e.g. 115111 (SEQ ID NO:29)) may be effective in refractory ornon-responding treatment resistance settings, e.g., tumors resistant totherapies involving tyrosine kinase inhibitors, such as, e.g.,lapatinib, and/or neratinib.

2. Cytotoxic Activities in the Presence of Other Drugs A. Lapatinib andT-DM1

The cytotoxicity of 115111 (SEQ ID NO:29) to HER2 positive cells wasevaluated in combination with lapatinib or T-DM1 using a cytotoxicityassay performed essential as described above. The cytotoxicities of115111 (SEQ ID NO:29) over range of concentrations combined with either1 µM lapatinib or a range of T-DM1 concentrations are shown in FIGS. 14and 15 . Neither combination appeared deleterious to the cytotoxicitycaused by 115111 (SEQ ID NO:29) to HER2 positive cells observed in thisin vitro cell-kill assay. Furthermore, the results of these experimentsshow the potential of combinations of 115111 (SEQ ID NO:29) with otheragents to achieve even more potent cytotoxicity to HER2-expressingcells.

FIG. 14 shows the results of cytotoxicity assays using HER2 positiveHCC1419 or HCC1954 cells where 115111 (SEQ ID NO:29), lapatinib, or acombination of both were administered to the cells. Concentrations for115111 (SEQ ID NO:29) and lapatinib were selected such that the singleagent resulted in about 50% viability for each cell line. The results ofthis experiment showed that 115111 (SEQ ID NO:29) may be administeredwith lapatinib to achieve more cytotoxicity than either administratedindividually. The data in FIG. 14 showed that a higher percentage ofcells were killed by the combination 115111 (SEQ ID NO:29) and lapatinibthan by treatment with either lapatinib or 115111 (SEQ ID NO:29) alone.These results showed that 115111 (SEQ ID NO:29) may be combined withlaptinib and/or may be administered in the presence of laptinib withoutsignificant loss in 115111 (SEQ ID NO:29) cell-kill activity to HER2positive cells. Furthermore, these results suggested that theadministration of the combination of 115111 (SEQ ID NO:29) (or a similarHER2-targeting molecule of the present invention) and lapatinib may killmore HER2 positive cells than using either one alone at the samerespective dose.

FIG. 15 shows the results of a cytotoxicity assay using HER2 positiveHCC1954 cells where 115111 (SEQ ID NO:29), T-DM1, or a combination ofboth were administered to the cells. The data in FIG. 15 showed that ahigher percentage of cells were killed by the combination 115111 (SEQ IDNO:29) and T-DM1 than by treatment with either T-DM1 or 115111 (SEQ IDNO:29) alone. These results showed that 115111 (SEQ ID NO:29) may becombined with T-DM1 and/or may be administered in the presence of T-DM1without significant loss in 115111 (SEQ ID NO:29) cell-kill activity toHER2 positive cells. Furthermore, these results suggested that theadministration of the combination of 115111 (SEQ ID NO:29) (or a similarHER2-targeting molecule of the present invention) and T-DM1 may killmore HER2 positive cells than using either one alone at the samerespective dose.

B. T-DM1 and Trastuzumab

The cytotoxic activities of 114912 (SEQ ID NO:28) and 115111 (SEQ IDNO:29) to HER2 positive HCC1954 cells were evaluated in the presence ofexcess trastuzumab using a cytotoxicity assay performed essentially asdescribed above, except that the cells were pre-treated with trastuzumab(20 µg/mL) for one hour prior to addition of the other HER2-targetingmolecules. FIG. 16 shows the results of cytotoxicity assays using HER2positive HCC1954 cells where 114912 (SEQ ID NO:28) and 115111 (SEQ IDNO:29) cytotoxic activities were evaluated in comparison to T-DM1, bothin the absence of trastuzumab and in the presence of excess trastuzumab.The pretreatment of HER2 positive cancer cells with excess trastuzumabdid not alter the cytotoxic activity of 115111 (SEQ ID NO:29) (see FIG.16 , middle). This was in contrast to the combination of excesstrastuzumab with T-DM1 or the exemplary HER2-targeting molecule 114912(SEQ ID NO:28), both of which comprise the heavy and light variabledomains of the trastuzumab antigen binding regions. These data suggestthat HER2-targeting molecules of the present invention may kill cells inthe presence of other HER2-binding therapeutics which bindnon-overlapping epitopes and do not compete or interfere with theHER2-targeting molecule’s mechanism of action (see FIG. 11 , trastuzumabbinds to an epitope mapped to domain IV of the ECD whereas 115111 (SEQID NO:29) interacts with domain I of the ECD). The monoclonal antibodytrastuzumab exhibited no cytotoxicity in this in vitro cytotoxic assay(involving just HER2 positive cancer cells and culture medium).

C. Trastuzumab and Pertuzumab

The cytotoxic activities of 115111 (SEQ ID NO:29) to HER2 positive cellsover a range of concentrations were evaluated in the presence of excesstrastuzumab, excess pertuzumab, or an excess of both trastuzumab andpertuzumab using a cytotoxicity assay performed essentially as describedabove, except that the cells were pre-treated with trastuzumab (100µg/mL), pertuzumab (100 µg/mL) or both trastuzumab (100 µg/ml) andpertuzumab (100 µg/mL) (for a total of 200 µg/mL antibody) for one hourprior to addition of the other HER2-targeting molecules. FIG. 17 andTable 8 show the results of the cytotoxicity assay using HER2 positiveHCC1954 or NCI-N87 cells where 115111 (SEQ ID NO:29) activity wasevaluated in the presence of excess trastuzumab, pertuzumab, or both.The HER2-targeting fusion protein 115111 (SEQ ID NO:29) killed HCC1954cells in the presence of excess trastuzumab (FIGS. 16-17 ). 115111 (SEQID NO:29) killed HCC1954 cells and NCI-N87 cells in the presence of bothexcess trastuzumab and excess pertuzumab or in the excess of both (FIG.17 ). The monoclonal antibodies trastuzumab or pertuzumab exhibited nocell-killing activity in this in vitro cytotoxic assay (involving justthe cancer cells and culture medium).

TABLE 8 Cytotoxic Activities of 115111 in the Presence of ExcessTrastuzumab, Pertuzumab, or both Trastuzumab and Pertuzumab TestCondition CD₅₀ (ng/mL) HCC-1954 NCI-N87 115111 alone 3.9 19.2 115111 +trastuzumab 5.4 44.0 115111 + pertuzumab 18.0 140.0 115111 + trastuzumaband pertuzumab 16.4 89.7

The data in FIG. 17 and Table 8 showed that the HER2-targeting molecule115111 (SEQ ID NO:29), tested at concentrations up to 20 µg/mL, wascytotoxic to HER2-expressing cells in the presence of excess trastuzumabor pertuzumab (either 100 µg/mL individually or 100 µg/mL of each, for atotal of 200 µg/mL concurrent exposure) thereby suggesting a potentialcombination therapy involving administering 115111 (SEQ ID NO:29) andanother HER-targeting therapeutic, such as a monoclonal antibody liketrastuzumab or pertuzumab having a non-overlapping HER2 binding epitope(see e.g. FIG. 11 ). Thus, the exemplary HER2-targeting molecule 115111(SEQ ID NO:29) has the potential to be effective in killing HER2expressing cancer cells in combination with other HER2-targetedtherapies as long as their HER2 binding epitopes are different.

The mechanism of action of the HER2-targeting molecules of the presentinvention -internalization and targeted ribosome inactivation- isdifferent than that of monoclonal antibodies targeting HER2 (trastuzumabbinding HER2 induces ADCC and inhibits HER2 signaling) (pertuzumabbinding HER2 inhibits dimerization) and is different than antibody drugconjugates like T-DM1 (targeted microtubule inhibition). Cancertherapies relying on drugs using mechanisms of action other than theone(s) used by HER2-targeting molecules may be rendered less effectivedue to the development of resistance mechanisms specific to one or moreof these other mechanisms of action. For example, tumors may becomeresistant to therapies involving monoclonal antibodies which bindspecific HER2 epitopes, such as, e.g., T-DM1, trastuzumab, and/orpertuzumab, and/or involving tyrosine kinase inhibitors, such as, e.g.,lapatinib and/or neratinib. Certain HER2-targeting molecules of thepresent invention (e.g. 115111 (SEQ ID NO:29)) may be effective inrefractory or non-responding treatment resistance settings, e.g., tumorsresistant to therapies involving T-DM1, trastuzumab, pertuzumab,lapatinib, and/or neratinib. Furthermore, the unique mechanism of actionof HER2-targeting molecules of the present invention may offer newopportunities for monotherapies for benefit-treatmentresistant/non-responder patients as well as for combination therapiescombining different and/or complementary mechanisms of action, such ascombinations with T-DM1, trastuzumab, pertuzumab, lapatinib, and/orneratinib.

3. Cytotoxicities of Exemplary HER2-Targeting Molecules Based onExposure Duration

To study the cytotoxic effects of exemplary HER2-targeting molecules ofthe present invention, kinetic cell-kill experiments were performed. Thecell-kill assay described above was used here but with differentdurations of exposure of the HER2-expressing cells to HER2-targetingmolecules. In this study, HER2 positive SKBR3 or HCC1954 were exposed toa HER2-targeting molecule for a specific and short duration of time(e.g. 1 or 4 hours), then the cells were washed and the media replaced(“washout”). Control samples were incubated with the HER2-targetingmolecule continuously throughout the experiment (with “no washing”). Alldata was normalized by comparing results to samples treated with vehiclealone under the same conditions. Results from this study are shown inTables 9-10 and FIGS. 18-19 .

TABLE 9 Cytotoxicity of Exemplary HER2-Targeting Molecules to HER2Positive SKBR3 Cells after Different Durations of Exposure SKBR3 TestCondition CD₅₀ (ng/mL) Fold change from continuous 114912 continuous44.6 N/A 114912 4-hour exposure 430.1 9.6 114912 1-hour exposure 3736.083.7 115111 continuous 11.1 N/A 115111 4-hour exposure 25.0 2.2 1151111-hour exposure 118.3 10.6 ^(∗) ‘N/A’ denotes not applicable

TABLE 10 Cytotoxicity of HER2 Positive HCC1954 Cells Under DifferentDuration of Exposure HCC-1954 Test Condition CD₅₀ (ng/mL) Fold changefrom continuous Cell viability at 2 µg/mL (%) Experiment 1 115111continuous 37.3 4.5% 115111 4-hour exposure 137.3 3.7 10.0% 114898continuous 110.1 9.6% 114898 4-hour exposure 487.4 4.4 44.9% Experiment2 115111 continuous 5.6 2.6% 115111 4-hour exposure 14.9 2.7 3.1% 115195continuous 3.9 3.5% 115195 4-hour exposure 14.3 3.7 3.5% 115645continuous 41.5 16.4% 115645 4-hour exposure 422.3 10.2 55% 115845continuous 25.3 16.2% 115845 4-hour exposure 179.4 7.1 45.2%

Under continuous exposure, 115111 (SEQ ID NO:29) exhibited a CD₅₀ valueof 5.6 ng/mL toward HER2 positive HCC1954 cells, which is similar to theCD₅₀ value of 3.9 ng/mL measured for 115195 (SEQ ID NO:32). Undercontinuous exposure, 115111 (SEQ ID NO:29) exhibited CD₅₀ values of 5.6and 37.3 ng/mL toward HCC1954 cells, which is similar to the CD₅₀ value25.3 ng/mL measured for 115845 (SEQ ID NO:35) and 41.5 ng/mL measuredfor 115645 (SEQ ID NO:34). However, the shorter exposure conditionrevealed some surprising differences between 115111 (SEQ ID NO:29) and115195 (SEQ ID NO:32) and the other HER2-targeting molecules tested.

The data from these experiments demonstrated that 115111 (SEQ ID NO:29)has potent cytotoxic activity when exposed to HER2-expressing cells forjust a short duration (1 to 4 hours) (see Tables 9-10 and FIGS. 18-19 ).The dimeric/divalent 115195 (SEQ ID NO:32) is related tomonomeric/monovalent 115111 (SEQ ID NO:29), differing only in a linker(which affects multimerization), exhibited similar activity to 115111(SEQ ID NO:29) under both continuous and washout conditions (Table 10,FIG. 19 ). In contrast, other HER2-targeting molecules like 114912 (SEQID NO:28), 114898 (SEQ ID NO:31), 115645 (SEQ ID NO:34), and 115845 (SEQID NO:35) exhibited greater reductions in cytotoxic activity at shorterexposure durations as compared to continuous exposure (see e.g. Tables9-10 and FIGS. 18-19 ).

The HER2 binding and catalytic activity data shown above did not providea clear indication that the cytotoxic potency under relatively shortdurations of exposure would be highest for 115111 (SEQ ID NO:29) and115195 (SEQ ID NO:32) as opposed to other HER2-targeting moleculestested, such as the trastuzumab based 114912 (SEQ ID NO:28).Furthermore, the cytotoxicity assay data above gathered under conditionsof continuous exposure (e.g. 3 to 5 days) did not provide any indicationas to which HER2-targeting molecules would be more potent underconditions of shorter exposure durations (e.g. 4 hours or less).

Comparing different HER2-targeting molecules, it is apparent that thebinding affinity does not always correlate with the cytotoxic activity.For example, 115645 (SEQ ID NO:34) and 115845 (SEQ ID NO:35) havesimilar cytotoxic activities toward HER2 positive HCC 1954 (Table 10;FIG. 19 ) but differ in HER2 binding as measured by Bmax (Table 5 andFIG. 10 ). Conversely, 115111 (SEQ ID NO:29) is more potently cytotoxicto HCC1954 cells than 115845 (SEQ ID NO:35) (see Tables 7-9; FIGS. 6-7and 18-19 ), but these two HER2-targeting molecules demonstrated similarBmax and K_(D) binding characteristics to those cells (see Tables 5 and10; FIG. 10 ). The shorter duration exposures reveal quite largedifferences between molecules, and this finding could not be predictedby the HER2 binding data, catalytic data, and the continuous exposuredata.

4. Species Cross-Reactivity of HER2 Binding

In preparation for animal studies, in vitro binding assays were used toinvestigate the binding of 115111 (SEQ ID NO:29) to recombinant HER2proteins from different species. The wells of enzyme-linkedimmunosorbent assay (ELISA) plates were coated with recombinant HER2extracellular domain (ECD) proteins derived from human (SEQ ID NO:38),cynomolgus monkey species (GenBank EHH58073.1 and NCBI referenceXP_001090430.1 (SEQ ID NOs: 40-41)), and mouse (UniProt P70424 (SEQ IDNO:42)) sources. The wells were blocked, washed, and then incubated withone of the exemplary HER2-targeting molecules: 115111 (SEQ ID NO:29),115195 (SEQ ID NO:32), or 114912 (SEQ ID NO:28). Unbound protein wasremoved by washing, and HER2 bound HER2-targeting molecules weredetected using a horse radish peroxidase (HRP)-conjugated mAbanti-SLTA-DI-2 that recognizes the Shiga toxin effector polypeptidecomponent in these molecules. The results of these binding assays arereported below (see Table 11 and FIG. 20 ).

TABLE 11 Binding to Recombinant HER2 Proteins from Different SpeciesHER2 ECD recombinant protein 115111 binding 115195 binding 114912binding K_(D) (ng/mL) Bmax (Abs 450 nM) K_(D) (ng/mL) Bmax (Abs 450 nM)K_(D) (ng/mL) Bmax (Abs 450 nM) human 19.11 4.00 12.30 3.96 44.80 3.57cynomolgus monkey 19.34 3.99 12.70 3.99 61.86 3.92

The data in Table 11 and FIG. 20 show that 115111 (SEQ ID NO:29), therelated molecule 115195 (SEQ ID NO:32) (which is a divalent dimervariant of the monovalent 115111 (SEQ ID NO:29) differing only in alinker), and the trastuzumab binding domain-derived molecule 114912 (SEQID NO:28) all bound to both human and cynomolgus monkey recombinant HER2ECD proteins (Sino Biological Inc., Beijing, CN, catalog nos. 10004-H02Hand 90295-C02H, respectively). In this assay, 114912 (SEQ ID NO:28) didnot bind the mouse HER2 ECD protein (Sino Biological Inc., Beijing, CN,catalog no. 50714-M02H). 115111 (SEQ ID NO:29) did not bind to the mouseHER2 ECD protein in this assay (FIG. 20 ). As shown in Table 11 and FIG.20 , the monomeric/monovalent 115111 (SEQ ID NO:29) and the relateddimeric/divalent 115195 (SEQ ID NO:32) exhibited similar binding tohuman and cynomolgus monkey recombinant HER2 ECD proteins. Although114912 (SEQ ID NO:28) bound with a similar affinity to human andcynomolgus monkey HER2 ECD proteins as 115111 (SEQ ID NO:29) and 115195(SEQ ID NO:32) regarding Bmax (see Table 11; FIG. 20 ), the trastuzumabbased 114912 (SEQ ID NO:28) exhibited a slightly higher K_(D) (2 to 5-fold) as compared to both 115111 (SEQ ID NO:29) and 115195 (SEQ IDNO:32) (see Table 11).

Summary of In Vitro Data:

Based on the in vitro data above, the most promising HER2-targetingmolecules tested were 114912 (SEQ ID NO:28), 115111 (SEQ ID NO:29),115195 (SEQ ID NO:32), 115172 (SEQ ID NO:23), 115194 (SEQ ID NO:33),115411 (SEQ ID NO:30), 115645 (SEQ ID NO:34), and 115845 (SEQ ID NO:35)(see Tables 1-3 and 9-10; FIGS. 3, 6-7, and 18-19 ). TheseHER2-targeting molecules exhibited potent cytotoxic activities in vitrounder conditions of continuous exposure. In the in vitro ribosomeinhibition assay, no significant differences were observed between theHER2-targeting molecules tested. In the in vitro cell-binding assay,some differences were observed between the HER2-targeting moleculestested; however, binding characteristics do not necessarily correlatewith cytotoxic potency (see above discussion regarding 115645 (SEQ IDNO:34) and 115845 (SEQ ID NO:35)). The in vitro cytotoxicity data abovegave no indication of which molecule(s) from among 115111 (SEQ IDNO:29), 115195 (SEQ ID NO:32), 115172 (SEQ ID NO:23), 115194 (SEQ IDNO:33), and 115411 (SEQ ID NO:30) would be the most effective and safein vivo. However, the Shiga toxin A Subunit effector polypeptidecomponents in 115172 (SEQ ID NO:23) and 115194 (SEQ ID NO:33) were lessde-immunized than the Shiga toxin A Subunit effector polypeptidecomponents of the other promising HER2-targeting molecules 115111 (SEQID NO:29), 115411 (SEQ ID NO:30), and 115195 (SEQ ID NO:32). Thus,administration of 115111 (SEQ ID NO:29), 115411 (SEQ ID NO:30), or115195 (SEQ ID NO:32) to animals was expected to be better toleratedthan administration of either 115172 (SEQ ID NO:23) or 115194 (SEQ IDNO:33). Animal studies involving the administration of differentexemplary HER2-targeting molecules at different dosages were undertaken,particularly to discern any differences in their safety profiles and/orefficacy for treating cancer and to pick the most promising candidate totake into human clinical trials. Neither the in vitro data above nor thefact that 115111 (SEQ ID NO:29) was monovalent whereas 115195 (SEQ IDNO:32) forms a dimeric divalent molecule suggested which one of thesetwo exemplary HER2-targeting molecules might perform better in vivo.

C. In Vivo Studies of Exemplary HER-Targeting Molecules of the PresentInvention

Animal models were used to investigate the in vivo effects of exemplaryHER-targeting molecules of the present invention. Different mousestrains were used to test the effect(s) of 115111 (SEQ ID NO:29) afteradministration on both healthy, immunocompetent mice and on xenografttumors in immunocompromised mice resulting from the injection into thosemice of human neoplastic cells which express HER2 on their cellsurfaces. Non-human primates were used to investigate the effect(s) of115111 (SEQ ID NO:29) after intravenous administration to healthynon-human primates. The exemplary HER2-targeting molecule 115111 (SEQ IDNO:29) was observed to be better tolerated than other HER2-targetingmolecules tested in animals. Surprisingly, 115111 (SEQ ID NO:29)administration was observed to be better tolerated than administrationof the closely related candidate HER2-targeting molecule 115195 (SEQ IDNO:32).

1. Serum Exposure

Immunocompetent C57BL/6 mice were used to investigate the serum exposure(pharmacokinetics) of 115111 (SEQ ID NO:29) and 115195 (SEQ ID NO:32)after intravenous (IV) administration of a single dose at 1 milligramper kilogram (mg/kg) of body weight. The HER2-targeting molecules 115111(SEQ ID NO:29) and 115195 (SEQ ID NO:32) were each diluted in phosphatebuffered saline (PBS) and administered by IV to mice, with three micewere per treatment group. At each timepoint, blood sera were collectedfrom each mouse, and the concentration of HER2-targeting molecule wasmeasured by means of a mesoscale discovery assay (Cambridge BiomedicalInc., Boston, MA, U.S.A.) using recombinant, human HER2 protein tocapture any HER2-binding molecules and using the anti-SLTA-DI-2 mAb forquantitation of HER2-targeting molecule present, both quantified usingstandard curves. Results from this study are shown in Table 12.

TABLE 12 Serum Exposure to Exemplary HER2-Targeting Molecules in C57BL/6Mice after Repeat Dosing Serum HER2-Targeting Molecule (ng/mL) Time (hr)115111 115195 0.083 22,344 30,280 0.5 3,603 29,899 1 1,354 23,334 2 35513,518 4 182 6,742 8 84 3,428 12 21 602 24 - 111

Data in Table 12 indicate that the serum exposure of 115195 (SEQ IDNO:32) was significantly longer than that of 115111 (SEQ ID NO:29) inmice.

2. Tolerability A. Tolerability in Immunocompetent Mice

Immunocompetent BALB/c mice were used to investigate the toxicity andimmunogenicity of exemplary HER2-targeting molecules 115111 (SEQ IDNO:29), 115172 (SEQ ID NO:23), 115195 (SEQ ID NO:32), and 115194 (SEQ IDNO:33) after repeated administrations over time to the same mice (6doses total per mouse). 115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23),115195(SEQ ID NO:32), and 115194 (SEQ ID NO:33) were diluted in PBS andadministered to mice (n = 6 per group at intravenous (IV) at doses 1mg/kg of body weight with dosing on days 1, 3, 5, 8, 10, and 12). As acontrol, vehicle-only samples were administered to a negative controltreatment group. The body weights and health of the mice were monitoredduring the study. Treatment tolerability results from this study areshown in Table 13 and FIG. 21 , with changes in body weight and deathsas indicators of tolerability. The percentage change in the mean bodyweight of the treatment group compared to Day 0 is shown along with thenumber of animal deaths per total animals in the treatment group.

TABLE 13 Effects of Exemplary HER2-Targeting Molecules in BALB/c Miceafter Repeat Dosing Sample Days dosed Amount per Dose Percentage BodyWeight Nadir (Day) Deaths / Number of Mice in Group Vehicle Control 1,3, 5, 8, 10, 12 N/A -2% (14) 0/6 115111 1, 3, 5, 8, 10, 12 1 -1% (12)0/6 115195 1, 3, 5, 8, 10, 12 1 -24% (14) ⅚ 115172 1, 3, 5, 8, 10, 12 1-9.5% (12) 0/6 115194 1, 3, 5, 8 (dosing halted) 1 -28% (11) 6/6 ^(∗)‘N/A’ denotes not applicable

The data from this study demonstrated that 115111 (SEQ ID NO:29) was themost well-tolerated molecule among the exemplary HER-targeting moleculestested (see Table 13 and FIG. 21 ). The group of mice that received115111 (SEQ ID NO:29) exhibit no loss in body weight compared to thecontrol group and an absence of animal deaths. By contrast, the group ofmice administered 115195 (SEQ ID NO:32) lost an average of over 20% bodyweight per mouse and 5 of 6 mice (83%) died during the first two weeksof the study. This indicates that the monomeric/monovalent 115111 (SEQID NO:29) was better tolerated than the related dimeric/divalentmolecule 115195 (SEQ ID NO:32). In addition, the 115111 (SEQ ID NO:29)molecule, comprising SLTA-DI-2 (SEQ ID NO:20), was better tolerated ascompared to 115172 (SEQ ID NO:23), which comprises the SLTA-FR (SEQ IDNO:37) component. This might be expected as 115111 (SEQ ID NO:29)comprises a more de-immunized Shiga toxin effector polypeptide componentcompared to 115172 (SEQ ID NO:23). The group of mice that received115172 (SEQ ID NO:23) had an average of 9.5% body weight loss and noanimal deaths with all the mice recovering body weight after dosing wascomplete. The least tolerated molecule in this study was 115194 (SEQ IDNO:33), which was dimeric/divalent and comprised the SLTA-FR Shiga toxineffector polypeptide component (SEQ ID NO:37). The group of mice thatreceived 115194 (SEQ ID NO:33) had the most body weight loss, resultingin a cessation of dosing after the 4^(th) dose; however, all the mice (6of 6 (100%)) in this dosing group died by Day 12 of the study.

Together, these data indicated that 115111 (SEQ ID NO:29) was the mostwell-tolerated HER2-targeting molecule in the study. Based on the serumexposure data for 115111 (SEQ ID NO:29) and 115195 (SEQ ID NO:32), onemight expect administration of 115195 (SEQ ID NO:32) to result in lessfree Shiga toxin in the serum and thus better tolerability. However,115195 (SEQ ID NO:32) resulted in more toxicity for reasons not yetfully elucidated.

Immunocompetent C57BL/6 mice were used to investigate the tolerabilityof 115111 (SEQ ID NO:29) after repeated administrations over time to thesame mice (2 cycles, 12 doses total per mouse). A 115111 (SEQ ID NO:29)sample was diluted in PBS and administered intravenously (IV) to mice (n= 6-10 mice per group) at doses of 0.1 mg/kg of body weight. As acontrol, the in vivo effects of the less de-immunized 115172 (SEQ IDNO:23) (see e.g. WO 2016/196344) and 115411 (SEQ ID NO:30) were studiedin parallel in the same study. As a control, vehicle-only samples wereadministered to a negative control treatment group. The body weights andhealth of the mice were monitored during the study and the results formice dosed at 1 mg/kg per dose are shown in Table 14 and FIG. 22 .

TABLE 14 Effects of Exemplary HER2-Targeting Molecules in C57BL/6 Miceafter Repeat Dosing Sample Days dosed Amount per Dose Body Weight Nadir% (Day) Deaths / Number of Mice in Group Vehicle Control 1, 3, 5, 8, 10,12; 22, 24, 26, 29, 31, 33 N/A -- 0/6 115111 1, 3, 5, 8, 10, 12; 22, 24,26, 29, 31, 33 1 -6.2% (11) 0/6 115172 1, 3, 5, 8, 10, 12, 22 (dosinghalted) 1 -14.5% (11) 6/6 115411 1, 3, 5, 8, 10, 12; 22, 24, 26, 29, 31,33 1 -10.2% (13) 0/6 ^(∗) ‘N/A’ denotes not applicable

The body weight and animal death results in Table 14 showed that 115111(SEQ ID NO:29) and 115411 (SEQ ID NO:30) were better tolerated than115172 (SEQ ID NO:23). None of the mice in the 115111 (SEQ ID NO:29) or115411 (SEQ ID NO:30) treatment groups died, whereas 6 of 6 mice (100%)died in the 115172 (SEQ ID NO:23) treatment group during the firsttwenty-two days of the study. The body weight data in FIG. 22 showedthat 115111 (SEQ ID NO:29) was better tolerated than 115172 (SEQ IDNO:23). These results indicate the 115111 (SEQ ID NO:29) and 115411 (SEQID NO:30) molecules, comprising SLTA-DI-2 (SEQ ID NO:20), were bettertolerated as compared to 115172 (SEQ ID NO:23), which comprised the lessde-immunized SLTA-FR Shiga toxin effector polypeptide component (SEQ IDNO:37).

B. Immunogenicity

Immunocompetent BALB/c mice were used to investigate theimmunogenicities of 115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), 115195(SEQ ID NO:32), and 115194 (SEQ ID NO:33) after repeated administrationsover time to the same mice (6 doses total per mouse). In one study,115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), 115195 (SEQ ID NO:32), and115194 (SEQ ID NO:33) were diluted in PBS and administered to mice (n =6 per group) at intravenous (IV) doses of 1 mg/kg of body weight. Immuneresponse results measured in this study are reported below and in FIG.23 . In another study, 115111 (SEQ ID NO:29), 115172 (SEQ ID NO:23), ora vehicle-only control sample were administered to mice (n = 6 pergroup) via intraperitoneal (IP) injection at doses of 0.25 mg/kg of bodyweight.

The quantities of anti-drug antibodies (ADA) to HER2-targeting moleculein the mice were monitored during these studies. The anti-drug antibodylevels were monitored by taking serum samples from the mice, dilutingthe samples, and incubating the diluted samples with the HER2-targetingmolecule being tested (115111 (SEQ ID NO:29) or 115172 (SEQ ID NO:23))overnight to form immune complexes. Then, molecules comprising a humanHER2 binding domain (e.g. a HER2-targeting molecule of the presentinvention) were captured using an ELISA method involving a plate coatedwith recombinant human HER2 protein. The quantity of immune complexespresent in each sample was then estimated using an anti-mouseimmunoglobulin G conjugated to horseradish peroxidase (IgG-HRP) toquantify the number of anti-mouse IgG’s captured by the human HER2 ELISAmethod. Results from this anti-drug antibody (ADA) assay are shown inFIG. 23 .

The top of FIG. 23 showed the more de-immunized 115111 (SEQ ID NO:29)resulted in a lower IgG anti-drug antibody (ADA) ELISA signal than115172 (SEQ ID NO:23) during the first 36 days of a study involvingintraperitoneal (IP) dosing at 0.25 mg/kg body weight. The bottom ofFIG. 23 showed administration of the more de-immunized 115111 (SEQ IDNO:29) resulted in a lower IgG ADA ELISA signal than administration of115172 (SEQ ID NO:23) at Day 22 of the study involving IV dosing at 1mg/kg body weight. These results are not unexpected as 115111 (SEQ IDNO:29) comprised a more de-immunized Shiga toxin effector polypeptidecomponent compared to 115172 (SEQ ID NO:23).

C. Non-Human Primates

Two toxicology studies were performed in cynomolgus monkeys involvingthe administration of 115111 (SEQ ID NO:29) in six intravenous (IV)bolus doses, three times per week for 2 weeks, to evaluate the potentialreversibility, progression or delayed progression of any findingsfollowing a 3-week (or 18 days for the low dose study) post-doseobservation period.

A “high dose” good laboratory practice (GLP) study tested doses of 50,150, and 300 µg/kg of body weight. These “high” doses were not welltolerated.

A “low dose” GLP study tested doses of 1, 5, and 25 µg/kg of bodyweight. These “low” doses showed effects that were not considered tohave adversely impacted the health of the animals and so theNo-Observed-Adverse-Effect Level (NOAEL) and Highest Non-Severely ToxicDose (HNSTD) for these two studies was 25 µg/kg body weight (i.e. thehighest dose tested in the “low-dose” study).

3. HER2 Expressing Tumor Xenograft Efficacy

A mouse model of human breast cancer was used to test the effect(s) of115111 (SEQ ID NO:29) after intraperitoneal (IP) administration. First,human neoplastic cells which express HER2 on their cell surfaces wereinjected into the mice and then the mice were treated with 115111 (SEQID NO:29).

The activity of 115111 (SEQ ID NO:29) was studied in a subcutaneoustumor model using SCID Beige mice bearing subcutaneous, human HER2positive tumors. HCC1954 cells from a human breast cancer derived cellline were implanted subcutaneously, then treatment was initiated whentumors reached an average of 100-150 mm³, thereby defining study Day 0.The mice were intravenously administered two cycles of 115111 (SEQ IDNO:29) at doses of 0.1, 0.5 or 2 mg/kg body weight on study days 1, 3,5, 8, 10, 12, 22, 24, 26, 29, 31 (and 33 for the groups administereddoses of 0.1 or 0.5 mg/kg). Tumor volume was monitored during thetreatment period and results are shown from Day 0 to Day 60 (latestpoint where greater than fifty percent of the animals in all groupssurvived) are shown in FIG. 24 . In addition, survival results from thestudy are shown from Day 0 to Day 84 (end of study) in FIG. 24 .

This study showed that repeat administration of 115111 (SEQ ID NO:29) at0.1, 0.5, and 2 mg/kg body weight resulted in delays in tumor growth andprovided survival benefits through study Day 84 (see e.g. FIG. 24 ).Weight losses observed in the 115111 (SEQ ID NO:29) treatment group thatwas administered doses of 2 mg/kg of body weight resulted in the lastscheduled dose (Day 33) to be withheld, and two mice in the 2 mg/kg115111 (SEQ ID NO:29) treatment group died during the study.

SUMMARY

Surprisingly, the exemplary HER2-targeting molecule 115111 (SEQ IDNO:29) was much safer to administer to animals at doses ranging between1 to 2 mg/kg body weight than any other exemplary HER2-targetingmolecules tested in this Example. This safety result could not bepredicted from the in vitro data above. It was particularly surprisingthat the monomer 115111 (SEQ ID NO:29) was better tolerated than thedimer 115195 (SEQ ID NO:32) because these two molecules share identicalcomponents: the Shiga toxin effector polypeptide, heavy variable chain,light variable chain, and linker between the Shiga toxin effectorpolypeptide and the HER2-binding region are the same in these molecules.Furthermore, the in vitro data revealed no significant differencebetween these molecules except that 115195 (SEQ ID NO:32) was dimericand divalent whereas 115111 (SEQ ID NO:29) was monomeric and monovalent.For example, both 115195 (SEQ ID NO:32) and 115111 (SEQ ID NO:29)exhibited similar cytotoxicity in vitro (see Tables 3 and 10; FIG. 19 )and similar binding characteristics to HER2 positive cells (see Tables 5and 11; FIG. 10 ). Both 115111 (SEQ ID NO:29) and 115195 (SEQ ID NO:32)maintained cytotoxic potency at shorter durations of exposure comparedto continuous exposure (see Tables 9-10 and FIGS. 18-19 ). 115111 (SEQID NO:29) exhibited more cytotoxic potency than 114912 (SEQ ID NO:28)(see Tables 2 and 9; and FIGS. 6 and 18 ), under conditions involvingcontinuous or shorter exposures. The HER2-targeting molecules 114912(SEQ ID NO:28) and 114778 (SEQ ID NO:24), both of which are partly basedon the widely used medicine trastuzumab (Herceptin®), were not among themost potent HER2-targeting molecules tested herein.

Example 2. Subject Response Prediction and Appropriate SubjectIdentification for a Therapeutic Use of a HER2-Targeting Molecule of thePresent Invention

Exemplary HER2-targeting molecules, such as one described above inExample 1, are administered to patient derived cell-lines in vitro inorder to identify the most sensitive or resistant histologies and/orhistological subtypes for different diseases, disorders, and/orconditions. Then, patients are categorized into different subpopulationsusing methods known to the skilled worker. For example, panels of breastcancer derived cell-lines (or alternatively gastric, ovary, lung,colorectal, melanoma, or pancreas) are tested in vitro using thecytotoxicity assay described in Example 1 or other methods known to theskilled worker. In addition, these cytotoxicity assays are performed incombination with an additional HER2-targeted therapeutic, such as, e.g.,including T-DM1 (trastuzumab emtansine), trastuzumab, and pertuzumab,and/or an additional drug which impinges on HER2 function, such as,e.g., lapatinib and/or neratinib. The cytotoxicity results revealcertain associations useful to identify or predict which indications,cell-types, histologies, histological subtypes, and/or subjects mayrespond or be resistant to a particular HER2-targeting molecule of thepresent invention and/or a particular combination of a HER2-targetingmolecule of the present invention and an additional therapeutic, such asanother HER2-targeted therapeutic described herein, based on informationregarding each cell line, such as, e.g., molecular markers (includingbiomarker expression, gene copy numbers, mutations and polymorphisms,pro-proliferation pathway activation or growth-inhibitor pathwayinhibition, metabolic activities, genotyping); responses to standardchemotherapeutic/cytotoxic agents, hormonal agents (e.g. estrogen,estradiol, etc.), and biologic agents (e.g. immune checkpointinhibitor); and abilities to grow on plastic and in soft agar, as wellas to grow in vivo subcutaneously and orthotopically. Further, malignanttissue specimens of cancers from individual patients or circulatingtumor cells are tested for the presence of biomarkers ofsensitivity/resistance using routine methods known to the skilledworker. Based on this biomarker associations, histologic associations,and cell characteristics in vitro, patients are selected or categorizedas appropriate candidates (or inappropriate) to receive HER2-targetingmolecule therapies (including combination therapies) as part of thetreatment regimen for their malignancy.

While some embodiments of the invention have been described by way ofillustration, it will be apparent that the invention may be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. The international pat. application publications WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, WO 2016/196344, WO2017/019623, and WO 2018/140427 are each incorporated herein byreference in its entirety. The disclosures of U.S. Pat. applicationsUS2015/259428, US2016/17784, US2017/143814, and US 62/659,116 are eachincorporated herein by reference in its entirety. The completedisclosures of all electronically available biological sequenceinformation from GenBank (National Center for Biotechnology Information,U.S.) for amino acid and nucleotide sequences cited herein are eachincorporated herein by reference in their entirety.

Sequence Listing ID Number Text Description Biological Sequence SEQ IDNO:1 Shiga-like toxin 1 Subunit A (SLT-1A)KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILM RRTISS SEQ ID NO:2 Shiga toxinSubunit A (StxA) KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAIL MRRTISS SEQ ID NO:3 Shiga-liketoxin 2 Subunit A (SLT-2A) DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKQKTECQIVGDRAAIKVNNVLWEANTIAALLNRKPQD LTEPNQ SEQ ID NO:4 Shiga toxinsubtype c Subunit A (Stx1cA) KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSVNAILGSVALILNCHHH ASRVAR SEQ ID NO:5 Shiga toxinsubtype d Subunit A (Stx1dA) KEFTLDFSTAKKYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDIMGLEPEEERFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTRAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSYSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSILPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASR VAR SEQ ID NO:6 Shiga toxinsubtype e Subunit A (Stx1eA) QDFTVDFSTAKKYVDSLNAIRSAIGTPLHSISSGGTSLLMIDNGTGDNLFAVDIRGLDPEEERFDNLRLIIERNNLYVTGFVNRTSNIFYRFADFSHVTFPGTRAVTLSGDSSYTTLQRVAGIGRTGMQINRHSLTTSYLDLMSYSGSSLTQPVARAMLRFVTVTAEALRFRQIQRGFRTTLDDVSGHSYTMTVEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGGVNAILGSVALILNCHHHT SRVSR SEQ ID NO:7 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 1REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARS VR SEQ ID NO:8 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 2REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFAHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARS VR SEQ ID NO:9 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 3REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDIYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:10 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 4REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPSVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:11 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 5REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYMAGFVNTATNTFYRFSDFTHISVPSVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:12 Shiga toxinsubtype 2c Subunit A (Stx2cA) variant 6REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQVLSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILSTVAVILNCHHQGARSV R SEQ ID NO: 13 Shiga toxinsubtype 2d Subunit A (Stx2dA) variant 1REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFAHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVIPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:14 Shiga toxinsubtype 2d Subunit A (Stx2dA) variant 2REFMIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPEEVDLTLNWGRISNVLPEFRGEGGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:15 Shiga toxinsubtype 2d Subunit A (Stx2dA) variant 3REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVIPEYRGEDGVRVGRISFNNISAILSTVAVILNCHHQGARSV R SEQ ID NO:16 Shiga toxinsubtype 2e Subunit A (Stx2eA) variant 1QEFTIDFSTQQSYVSSLNSIRTAISTPLEHISQGATSVSVINHTPPGSYISVGIRGLDVYQERFDHLRLIIERNNLYVAGFVNTTTNTFYRFSDFAHISLPGVTTISMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRLALSETAPVYTMTPEDVDLTLNWGRISNVLPEYRGEAGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:17 Shiga toxinsubtype 2e Subunit A (Stx2eA) variant 2QEFTIDFSTQQSYVSSLNSIRTAISTPLEHISQGATSVSVINHTPPGSYISVGIRGLDVYQAHFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFAHISLPGVTTISMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSGNTMTREASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPEDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSV R SEQ ID NO:18 Shiga toxinsubtype 2f Subunit A (Stx2fA)DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHVPGGNYISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNTFYRFSDFSHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVR SEQ ID NO:19 SLTA-DI-1AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH14AS AVAA SEQ ID NO:20 SLTA-DI-2KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH14AS AVAA SEQ ID NO:21 SLTA-DI-3REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH14AS AVAA SEQ ID NO:22 114773(SLTA-FR::scFv1) MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTOSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY WGQGTLVTVSSA SEQ ID NO:23 115172(SLTA-FR::scFv-6) MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIK SEQ ID NO:24 114778 (SLTA-DI-1::scFv-1)MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY WGQGTLVTVSSA SEQ ID NO:25 114795(SLTA-DI-1::scFv2) MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLLQSGAELKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYY CAAWDDSLSGWVFGGGTKLTVLA SEQID NO:26 114791 (SLTA-DI-1::scFv3)MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSDIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFT FGSGTKLEIK SEQ ID NO:27SLTA-DI-1::scFv4 MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYW GQGTLVTVSSA SEQ ID NO:28 114912(SLTA-DI-2::scFv-5) MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSSEQ ID NO:29 115111 (SLTA-DI-2::scFv-6)MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIK SEQ ID NO:30 115411 (SLTA-DI-3::scFv-7)MREFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASPSTPPTPSPSTPPASQVQLVQSGPEVKKPGASVKVSCKASGYPFTNYGMNWVRQAPGQGLEWMGWINTSTGESTFADDFKGRVTMTTDTSTSTTYMELRSLRPDDTAVYFCARWEVYHGYVPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASIGDRVTITCKASQDVYNAVAWYQQKPGEAPKLLVYSASSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQHFRTPFTFAPGTKLEIK SEQ ID NO:31 114898MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRT AAQGTDYWGQGTQVTVSSA SEQ IDNO:32 115195 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFT FGSGTKLEIK SEQ ID NO:33115194 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFT FGSGTKLEIK SEQ ID NO:34115645 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQ VTVSSA SEQ ID NO:35 115845MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTV SSHHHHHH SEQ ID NO:36SLTA-DI-2::scFv-8 MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAE DLAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO:37 SLTA-FR KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:38 HER2 fromHomo sapiens MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:39 predictedextracellular domain of HER2 from Homo sapiensTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPIN CTHSCVDLDDKGCPAEQRASPLT SEQ IDNO:40 HER2 from Macaca fascicularisMELAAWYRWGLLLALLPPGAAGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGNPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPVCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLRVFETLEEITGYLYISAWPDSLPDLSVLQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTRLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGTCQSCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGTGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPRAPSEGTGSDVFDGDLGMGAAKGLQSLPAHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPLPQEGPLSPARPTGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLAPRGGAAPQPHLPPAFSPAFDNLYYWDQDPSERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:41 HER2 from Macacamulatta MELAAWYRWGLLLALLPPGAAGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDLLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPVCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLRVFETLEEITGYLYISAWPDSLPDLSVLQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTRLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGTCQSCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGTGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPRAPSEGTGSDVFDGDLGMGAAKGLQSLPAHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPQEGPLSPARPTGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLAPRGGAAPQPHLPPAFSPAFDNLYYWDQDPSERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:42 HER2 from Musmusculus MELAAWCRWGFLLALLSPGAAGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPANASLSFLQDIQEVQGYMLIAHNRVKHVPLQRLRIVRGTQLFEDKYALAVLDNRDPLDNVTTAAPGRTPEGLRELQLRSLTEILKGGVLIRGNPQLCYQDMVLWKDVLRKNNQLAPVDMDTNRSRACPPCAPTCKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALITYNTDTFESMLNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPCAGVCYGLGMEHLRGARAITSDNIQEFAGCKKIFGSLAFLPESFDGNPSSGVAPLKPEHLQVFETLEEITGYLYISAWPESFQDLSVFQNLRVIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNTHLCFVNTVPWDQLFRNPHQALLHSGNRPEEACGLEGLVCNSLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVWKGLPREYVRGKHCLPCHPECQPQNSSETCYGSEADQCEACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCPAEQRASPVTFIIATVVGVLLFLIIVVVIGILIKRRRQKIRKYTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPSSPMDSTFYRSLLEDDDMGELVDAEEYLVPQQGFFSPDPALGTGSTAHRRHRSSSARSGGGELTLGLEPSEEEPPRSPLAPSEGAGSDVFDGDLAVGVTKGLQSLSPHDLSPLQRYSEDPTLPLPPETDGYVAPLACSPQPEYVNQPEVRPQSPLTPEGPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLAPRAGTASQPHPSPAFSPAFDNLYYWDQNSSEQGPPPSTFEGTPTAENPEYLGLDVPV SEQ ID NO:43 intein and chitinbinding domain CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQP HTSLAGWEPSNVPALWQLQ SEQ ID NO:44polyhistidine tag (6xHis) HHHHHH SEQ ID NO:45 vhCDR1 DTYIH SEQ ID NO:46vhCDR2 RIYPTNGYTRYADSVKG SEQ ID NO:47 vhCDR3 WGGDGFYAMDY SEQ ID NO:48vlCDR1 RASQDVNTAVA SEQ ID NO:49 vlCDR2 SASFLYS SEQ ID NO:50 vlCDR3QQHYTTPPT SEQ ID NO:51 vhCDR1 SYWIA SEQ ID NO:52 vhCDR2LIYPGDSDTKYSPSFQG SEQ ID NO:53 vhCDR3 HDVGYCSSSNCAKWPEYFQH SEQ ID NO:54vlCDR1 SGSSSNIGNNYVS SEQ ID NO:55 vlCDR2 SASYRYT SEQ ID NO:56 vlCDR3QQYYIYPYT SEQ ID NO:57 vhCDR1 NYGMN SEQ ID NO:58 vhCDR2WINTSTGESTFADDFKG SEQ ID NO:59 vhCDR3 WEVYHGYVPY SEQ ID NO:60 vlCDR1KASQDVYNAVA SEQ ID NO:61 vlCDR2 SASSRYT SEQ ID NO:62 vlCDR3 QQHFRTPFTSEQ ID NO:63 vhCDR1 DYTMD SEQ ID NO:64 vhCDR2 DVNPNSGGSIYNQRFKG SEQ IDNO:65 vhCDR3 NLGPSFYFDY SEQ ID NO:66 vlCDR1 KASQDVSIGVA SEQ ID NO:67vlCDR2 SASYRYT SEQ ID NO:68 vlCDR3 QQYYIYPYT SEQ ID NO:69 vhhCDR1 INTMGSEQ ID NO:70 vhhCDR2 LISSIGDTYYADSVKG SEQ ID NO:71 vhhCDR3 FRTAAQGTDYSEQ ID NO:72 vhhCDR1 SCGMG SEQ ID NO:73 vhhCDR2 RISGDGDTWHKESVKG SEQ IDNO:74 vhhCDR3 CYNLETY SEQ ID NO:75 SLT-1A-combo variant 1KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:76SLT-1A-combo variant 2 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHA SAVAA SEQ ID NO:77SLT-1A-combo variant 3 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHA SAVAA SEQ ID NO:78SLT-1A-combo variant 4 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASA VAA SEQ ID NO:79 SLT-1A-combovariant 5 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:80SLT-1A-combo variant 6 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:81SLT-1A-combo variant 7 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:82SLT-1A-combo variant 8 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAS AVAA SEQ ID NO:83SLT-1A-combo variant 9 KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLAALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHAS AVAA SEQ ID NO:84SLT-1A-combo variant 10 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHA SAVAA SEQ ID NO:85SLT-1A-combo variant 11 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHA SAVAA SEQ ID NO:86SLT-1A-combo variant 12 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPMVATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHA SAVAA SEQ ID NO:87SLT-1A-combo variant 13 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGILGDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHAS AVAA SEQ ID NO:88SLT-1A-combo variant 14 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHIL RFSVAHKASAVAA SEQ ID NO:89SLT-1A-combo variant 15 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHAR NLVPMVATVASAVAA SEQ ID NO:90linker 1 EFPKPSTPPGSSGGAP SEQ ID NO:91 linker 2 GGGGSGG SEQ ID NO:92linker 3 GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:93 linker 4 GSTSGSGKPGSGEGSSEQ ID NO:94 linker 5 GGGGS SEQ ID NO:95 linker 6 AHHSEDPSSKAPKAP SEQ IDNO:96 linker 7 GSTSGSGKPGSGEGSTKG SEQ ID NO:97 exemplary HER2-targetingmolecule # 1 AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG QGTLVTVSSA SEQ ID NO:98 exemplaryHER2-targeting molecule #2 AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLLQSGAELKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYY CAAWDDSLSGWVFGGGTKLTVLA SEQ IDNO:99 exemplary HER2-targeting molecule #3AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSDIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTF GSGTKLEIK SEQ ID NO:100exemplary HER2-targeting molecule #4AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQ GTLVTVSSA SEQ ID NO:101exemplary HER2-targeting molecule #5KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSSEQ ID NO:102 exemplary HER2-targeting molecule #6KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIK SEQ ID NO:103 exemplary HER2-targetingmolecule #7 REFTLDFSTARTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAASPSTPPTPSPSTPPASQVQLVQSGPEVKKPGASVKVSCKASGYPFTNYGMNWVRQAPGQGLEWMGWINTSTGESTFADDFKGRVTMTTDTSTSTTYMELRSLRPDDTAVYFCARWEVYHGYVPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASIGDRVTITCKASQDVYNAVAWYQQKPGEAPKLLVYSASSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQHFRTPFTFAPGTKLEIK SEQ ID NO:104 exemplary HER2-targetingmolecule #8 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAA QGTDYWGQGTQVTVSSA SEQ ID NO:105exemplary HER2-targeting molecule #9KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTF GSGTKLEIK SEQ ID NO:106exemplary HER2-targeting molecule #10KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVT VSSA SEQ ID NO:107 exemplaryHER2-targeting molecule #11 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVS S SEQ ID NO:108 exemplaryHER2-targeting molecule #12 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAED LAVYFCQQHFRTPFTFGSGTKLEIKSEQ ID NO:179 (G4S)7 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:180(G4S)3 GGGGSGGGGSGGGGS SEQ ID NO:181 linker 8 SPSTPPTPSPSTPPA

1. A method of treating a disease, disorder, or condition involvingHER2-expressing cells in a patient, the method comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of a monovalent, monomeric HER2-targeting molecule comprising thepolypeptide sequence of SEQ ID NO: 29 or SEQ ID NO:
 102. 2. The methodof claim 1, wherein the polypeptide sequence consists of SEQ ID NO: 29or SEQ ID NO:
 102. 3. The method of claim 1 or claim 2, wherein themonovalent, monomeric HER2-targeting molecule is in the form of apharmaceutically acceptable solvate or salt.
 4. The method of any one ofclaims 1-3, wherein the monovalent, monomeric HER2-targeting molecule isadministered with at least one pharmaceutically acceptable excipient orcarrier.
 5. The method of any one of claims 1-4, wherein the methodfurther comprises administering to the patient in need thereof atherapeutically effective amount of an additional HER2-targetingtherapeutic agent.
 6. The method of any one of claims 1-5, wherein thepatient has been previously treated with at least one otherHER2-targeting therapeutic agent.